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Trademarks
Patented or patent-pending technology and/or registered or registration-pending trademark of QIAGEN: QIAGEN, QIAamp ® , QIAEX ® , QIAquick ® ,
DNeasy ® , HotStarTaq ™ , Oligotex ® , Omniscript ™ , RNeasy ® , Sensiscript ™ . iCycler is a trademark of Bio-Rad Laboratories, Inc.
Lightcycler is a registered trademark of Idaho Technology Inc.
RNAimage is a registered trademark of GenHunter Corporation.
SAGE is trademark of Genzyme Molecular Oncology.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
The PCR Process is covered by U.S. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG.
Differential display technology is covered by US patent No. 5,262,311 and other pending patents licensed to GenHunter Corporation. The SAGE process is covered by U.S. Patent 5,695,937 owned by Genzyme Molecular Oncology.
Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.
Notice to Purchasers: Limited License
A license held under U.S. Patents 4,683,202, 4,683,195, 4,965,188, and 5,075,216 or their foreign counterparts, owned by Hoffmann-La Roche
Inc. and F. Hoffmann-La Roche Ltd., has an up-front fee component and a running-royalty component. The purchase price of products containing
QIAGEN Taq DNA Polymerase or HotStarTaq DNA Polymerase includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction (PCR) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. These rights under the up-front fee component may be purchased from Perkin-Elmer Corporation or obtained by purchasing an authorized thermal cycler. No right to perform or offer commercial services of any kind using PCR, including, without limitation, reporting the results of purchaser’s activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Perkin-Elmer Corporation, 850 Lincoln Center Drive, Foster City, California 94404 or at Roche Molecular
Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.
© 2000 QIAGEN, all rights reserved.
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Taq DNA Polymerase
HotStarTaq DNA Polymerase
Taq and HotStarTaq Master Mix Kits
Omniscript and Sensiscript RT Kits
QIAGEN OneStep RT-PCR Kit
Standard PCR
Hot-start PCR
PCR screening
Single-cell PCR
GC-rich and other difficult templates
Longer PCR fragments
Multiplex PCR
Paraffin-embedded tissues
Methylation-specific PCR
RAPD-PCR
RT-PCR, two-step
RT-PCR, one-step
Single-cell RT-PCR
Differential display
SAGE analysis
31
36
38
40
42
11
26
28
30
20
22
23
11
13
15
19
9
9
10
4
5
5
7
8
Standard primers — design and use
Degenerate primers — design and use
Longer PCR fragments
Single-cell PCR
Multiplex PCR
Nested PCR
43
One-step RT-PCR primers — design and use
Multiplex RT-PCR
Co-amplification of an internal control
Longer RT-PCR products
49
51
52
53
46
47
48
43
44
45
Selecting the best sample preparation method 54
Optimal PCR product purification 55
56
57
Contents
QIAGEN PCR and RT-PCR Application Guide
3

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The Nobel prize-winning invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine.
Not only are PCR techniques now essential for many common procedures, but also new techniques and applications are constantly being developed.
PCR is now used all over the world in a staggering number of applications. In this guide, we survey a wide range of PCR applications, ranging from standard PCR and RT-PCR to more specialized PCR-based techniques, such as methylationspecific PCR, RAPD-PCR, differential display, and SAGE ™ analysis. Included are examples and guidelines for PCR and
RT-PCR from single cells, paraffin-embedded tissues, and difficult GC-rich templates, as well as challenging PCR techniques such as multiplex PCR and amplification of long
PCR fragments.
From Japan to Switzerland, from France to the USA, researchers worldwide rely on QIAGEN ® enzymes for highly sensitive and specific reverse transcription and PCR. This guide presents a spectrum of the research currently being carried out. We extend our thanks to those who have contributed to this project and hope that it may provide a useful guide to successful PCR and RT-PCR for researchers everywhere.
USA
Canada
United
Kingdom
France
Germany
Switzerland
Japan
4 Introduction
QIAGEN PCR and RT-PCR Application Guide

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Taq
QIAGEN Taq DNA Polymerase, used in combination with
QIAGEN PCR Buffer provides robust performance for reproducible results in a wide range of PCR applications without the need for time-consuming optimization. The innovative
PCR buffer has been developed to dramatically reduce the need for optimization of individual primer–template systems, saving time and effort.
● QIAGEN PCR Buffer for minimal PCR optimization
● Q-Solution, an innovative PCR additive, for amplification of difficult templates
● Choice of formats for convenience and ease of handling
QIAGEN Taq DNA Polymerase is a high-quality recombinant enzyme produced by QIAGEN and sold under a licensing agreement with Hoffmann-La Roche. This enzyme is suitable for standard PCR applications (see page 11) and specialized applications such as differential display (see page 40), PCRbased DNA fingerprinting (see page 30), RAPD-PCR (see page
30), and amplification of long PCR products (see page 22).
QIAGEN PCR Buffer has been developed to save time and effort by reducing the need for PCR optimization. During the annealing step of every PCR cycle, the buffer allows a high ratio of specific-to-nonspecific primer binding. Owing to a uniquely balanced combination of KCl and (NH
4
)
2
SO
4
, the
PCR buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg 2+ concentrations than do conventional PCR buffers (Figure 1).
Optimization of PCR by varying the annealing temperature or the Mg 2+ concentration is dramatically reduced and often not required. This makes challenging PCR possible, such as with
DNA from paraffin-embedded tissues (see page 26).
Wide Annealing-Temperature Window
A QIAGEN
M 50 52 54 56 58 60
Supplier P
II
50 52 54 56 58 60°C
– 1.5 kb
Tolerance to Variable Mg 2+ Concentration
B
M
QIAGEN
1.5 2.0 2.5 3.0 3.5 4.0
M
Supplier P
II
1.5 2.0 2.5 3.0 3.5 4.0 mM
– 0.75 kb
Figure 1
PCR amplification at the indicated annealing temperatures
( QIAGEN ). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier ( Supplier P
II
). Amplification of
■ the single-copy human cystic fibrosis gene and
■ and Mg 2+ concentrations
■ using QIAGEN PCR Buffer and Taq DNA Polymerase
■ the single-copy human prion protein gene. M : markers.
Taq DNA Polymerase
QIAGEN PCR and RT-PCR Application Guide
5

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Q-Solution is an innovative additive that facilitates amplification of difficult templates by modifying the melting behavior of nucleic acids. Q-Solution will often enable or improve suboptimal PCR systems caused, for example, by templates that have a high degree of secondary structure or that are GC-rich
(see page 20). Unlike other commonly used additives such as
DMSO, Q-Solution is used at just one working concentration.
Q-Solution can have varying effects, depending on the individual PCR assay. Q-Solution can enable an amplification that previously failed (Figure 2A) or increase specificity in certain primer–template systems (Figure 2B). In some cases,
Q-Solution has no effect on PCR performance (Figure 2C).
Addition of Q-Solution can, however, also cause reduced efficiency of a previously successful amplification reaction
(Figure 2D). In this latter case, addition of Q-Solution disturbs the previously optimal primer–template annealing. Therefore, when using Q-Solution for the first time for a particular primer–template system, it is always advisable to perform reactions in parallel with and without Q-Solution.
A M – – + + B M – – + +
– 2 kb
– 4.8 kb
C
M – – + + D M – – + +
– 0.5 kb
Figure 2
Q-Solution has different effects, depending on the primer–template system.
– : without Q-Solution;
+ : with Q-Solution; M : markers.
6 Taq DNA Polymerase
QIAGEN PCR and RT-PCR Application Guide
– 1.5 kb

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™
HotStarTaq ™ DNA Polymerase has been developed by
QIAGEN to provide hot-start PCR for higher PCR specificity.
The combination of HotStarTaq DNA Polymerase and the unique QIAGEN PCR Buffer minimizes nonspecific amplification products, primer–dimers, and background. It is ideal for amplification reactions involving complex genomic or cDNA templates, very low-copy targets, or multiple primer pairs. HotStarTaq DNA Polymerase makes hot-start PCR simple and easy, eliminating the extra handling steps and contamination risks associated with conventional hot-start methods.
● Higher PCR specificity
● Reduced nonspecific amplification
● Easy handling
HotStarTaq DNA Polymerase is a chemically modified form of the recombinant 94-kDa Taq DNA Polymerase from
QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer–dimers at low temperatures. HotStarTaq DNA
Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product (Figure 3). PCR setup is quick and convenient as all reaction components can be combined at room temperature. The high PCR specificity of HotStarTaq
DNA Polymerase makes it ideal for highly sensitive PCR applications such as pathogen detection (see page 13) and single-cell PCR (see page 19) as well as specialized applications such as SAGE ™ analysis (see page 42).
QIAGEN PCR Buffer supplied with HotStarTaq DNA
Polymerase provides the advantage of a balanced combination of cations to ensure specific primer annealing without optimization (see page 5 for details). This provides efficient and sensitive PCR even for specialized applications such as methylation-specific PCR (see page 28).
Q-Solution is an innovative additive that facilitates amplification of difficult templates by modifying the melting behavior of nucleic acids (see page 6 for details).
M
Superior Performance in Hot-Start PCR
H ot
St ar
Ta q
(Q
IA
G
EN
) e
H ot
-s ta rt
e nz ym
(S up pli er
P
)
II
A nt ib od ym
(S up ed ia te d pli er
L
)
N o ho t s ta rt
(S up pl ie r
R)
Figure 3
A 497-bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from
QIAGEN ( HotStarTaq ); hot-start enzyme from Supplier P
II
( Hot-start enzyme ); Taq–antibody mixture from Supplier L ( Antibody-mediated ); no hot start with enzyme from Supplier R ( No hot start ). Arrow indicates the specific PCR product. Equal volumes of the reaction were analyzed on a 2% agarose gel. M : markers.
➞
HotStarTaq DNA Polymerase
QIAGEN PCR and RT-PCR Application Guide
7

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Taq
Taq and HotStarTaq Master Mix Kits provide QIAGEN Taq and HotStarTaq DNA Polymerases in a premixed format.
These ready-to-use solutions include QIAGEN Taq or
HotStarTaq DNA Polymerase, QIAGEN PCR Buffer, and ultrapure dNTPs at optimized concentrations. Setting up amplification reactions is fast and easy — simply pipet the master mix into each PCR tube, and add an equal volume of primers and template DNA diluted in the distilled water provided with the kit (Figure 4). Pipetting steps are minimized, reducing the possibility of errors and contamination.
The combination of high specificity and easy handling makes the Taq and HotStarTaq Master Mix Kits ideal for use with complex genomic or cDNA templates, multiple primer pairs, templates isolated from difficult sources, and projects in which large numbers of samples are amplified, such as genetic screening and DNA fingerprinting (see page 15).
Taq DNA polymerase
10x buffer dNTPs Primers
Taq or HotStarTaq
Master Mix
Thaw
Calculate
Master mix Distribute
Distribute
Add template
Amplification
Figure 4
Standard PCR setup and PCR setup with Taq or HotStarTaq Master Mix Kit.
Add primers and template
Amplification
8 Taq and HotStarTaq Master Mix Kits
QIAGEN PCR and RT-PCR Application Guide

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™
™
QIAGEN offers two novel recombinant reverse transcriptases with high affinity for RNA. This high affinity results in highly specific and sensitive RT-PCR (Figure 5) even with low-copy transcripts and gives them the ability to read through even complex RNA secondary structures without adjusting temperature or reaction conditions. Regions of RNA with high GC content can cause other reverse transcriptases to stop, dissociate from the RNA template, or skip over looped-out regions of RNA.
These difficult templates, however, prove no problem for
QIAGEN reverse transcriptases. This results in an exceptionally high level of specificity, providing highly sensitive two-step
RT-PCR in applications such as gene-expression analysis and detection of viral pathogens (see pages 31–35).
● Novel RT enzymes and unique buffers for high specificity
● Detect as few as 10 copies or start with as little as 1 pg starting RNA
● High yields of cDNA
● Robust systems guarantee consistent results
Omniscript ™ Reverse Transcriptase has a wide dynamic range for sensitive and specific reverse transcription using any amount of RNA from 50 ng to 2 µg per reaction.
Sensiscript ™ Reverse Transcriptase was specially developed for very small amounts of RNA (<50 ng, including any carrier
RNA). This makes it especially suitable for RT-PCR from single cells (see Figure 6 and page 38) or small biopsies, as well as sensitive applications such as differential display (see page 40).
Superior Sensitivity and Dynamic Range of Omniscript RT
1000 500 250 125 62
NFκ B
Inositol receptor
31 ng RNA
Omniscript
(QIAGEN)
MMLV RNase H-
(Supplier L)
MMLV
(Supplier L)
AMV RNase H-
(Supplier L)
AMV
(Supplier P)
Figure 5
Reverse transcription was carried out with different reverse transcriptases according to suppliers’ specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA
Polymerase. A 1.7 kb β -actin fragment was amplified.
RT-PCR with RNA Corresponding to 1 Cell
β -Actin
500 50 10 1 0 cells bp
– 210
– 460
– 240
Dystrophin – 360
Figure 6
Total RNA was isolated from 10 to 5000 HeLa cells using the RNeasy
96 Kit. 1/10 of each eluate (corresponding to RNA from 1 to 500 cells) was used for reverse transcription with Sensiscript Reverse Transcriptase.
1/2 of each RT reaction was then used in a 40-cycle PCR with QIAGEN
Taq DNA Polymerase and primers specific for genes encoding β -actin,
NFκ B, inositol-1,4,5-triphosphate receptor, or dystrophin. Sizes of the amplicons are as indicated.
Omniscript and Sensiscript RT Kits
QIAGEN PCR and RT-PCR Application Guide
9

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The QIAGEN OneStep RT-PCR Kit provides a convenient format for highly efficient and specific RT-PCR using gene-specific primers and any RNA template. The kit contains optimized components that allow both reverse transcription and PCR amplification to take place in what is commonly referred to as a ”one-step” reaction.
●
●
●
Easy one-tube setup
One-step RT-PCR of any RNA template without optimization
● Unique blend of Omniscript and Sensiscript RTs and
HotStarTaq DNA Polymerase for high specificity and sensitivity
Optimized buffer for efficient reverse transcription and amplification
QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend for both reverse transcription and
PCR amplification. The high affinity and wide dynamic range of Omniscript and Sensiscript Reverse Transcriptases in the enzyme mix ensures highly efficient and sensitive reverse transcription of any RNA amount from 1 pg to 2 µg. In the same enzyme mix, HotStarTaq DNA Polymerase provides all the advantages of a hot start for PCR without compromising reverse transcription. During reverse transcription, HotStarTaq
DNA Polymerase (see page 7) is completely inactive and does not interfere with the RT reaction. A simple heating step simultaneously switches on the polymerase activity and switches off reverse transcription, inactivating the reverse transcriptases. This hot start eliminates nonspecific amplification products commonly associated with amplification from singlestranded DNA.
QIAGEN OneStep RT-PCR Buffer * is designed to enable both efficient reverse transcription and specific amplification. The unique buffer composition allows reverse transcription to be performed at higher temperatures (50°C). In combination with the high-affinity reverse transcriptases, this allows highly efficient reverse transcription by disrupting secondary structures and is particularly important for one-step RT-PCR with limiting amounts of template RNA (e.g., single-cell
RT-PCR, see page 38). As with all QIAGEN PCR buffers, the
QIAGEN OneStep RT-PCR Buffer also provides the advantage of a uniquely balanced combination of KCl and (NH
4
)
2
SO
4
.
This provides stringent primer-annealing conditions over a wider range of conditions than do conventional one-step
RT-PCR buffers and ensures specific primer annealing without optimization. The combination of high-affinity enzymes and optimized RT-PCR buffer provides highly sensitive RT-PCR in a wide variety of applications, such as gene-expression analysis, viral detection, multiplex, and single-cell RT-PCR (see page 36).
* Patent pending
Primers
QIAGEN
OneStep RT-PCR Kit:
Enzyme Mix
Buffer dNTP Mix
RNase-free water
Master mix
Distribute
Add template
10 QIAGEN OneStep RT-PCR Kit
QIAGEN PCR and RT-PCR Application Guide
RT-PCR
Figure 7
RT-PCR setup with the QIAGEN OneStep RT-PCR Kit.

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Standard PCR is the amplification of genomic or plasmid
DNA using a standard Taq DNA polymerase without special additives. Even with standard or routine applications, optimization of conditions is often necessary. It is important to use a PCR system combining a high-quality polymerase and a buffer system that minimizes optimization of conditions, such as temperature and Mg 2+ concentration, to save time and effort. In these standard applications, QIAGEN Taq
DNA Polymerase, used in combination with QIAGEN PCR
Buffer, provides robust performance for reproducible results in a wide range of PCR applications without the need for timeconsuming optimization (see page 5).
In this section, we present two examples of characterizing specific gene structures (genotyping) using PCR with
QIAGEN Taq DNA Polymerase.
1 2 3
The CYP2C19 gene encodes a cytochrome P-450 involved in metabolism of a number of clinically important drugs. Mutations in this gene can lead to poor drug metabolism. Genomic DNA from five individuals was analyzed for the presence of two mutations that differ from the wild-type CYP2C19*1 allele: the CYP2C19*2 mutation leads to a splicing error, and CYP2C19*3 is a nonsense mutation.
PCR using QIAGEN Taq DNA Polymerase or
HotStarTaq™ DNA Polymerase, followed by restriction digest, allowed accurate genotyping ( Figure 8 ).
Genotype
CYP2C19 *2/*2 *1/*2 *1/*3
4
*3/*3
5
*1/*1
329 bp
169 bp
Figure 8
Genomic DNA from five individuals was analyzed by PCR using QIAGEN Taq
DNA Polymerase or HotStarTaq DNA Polymerase. PCR was carried out for
35 cycles using primers specific for a 329 bp fragment of the CYP2C19 gene with the CYP2C19*2 mutation (left) or a 169 bp fragment with the CYP2C19*3 mutation (right). Following PCR, the CYP2C19*2 amplicon was digested with
Sma I (left) and the CYP2C19*3 amplicon with Bam HI (right). The wild-type
( CYP2C19*1 ) allele contains both restriction sites. Mutant alleles CYP2C19*2 and CYP2C19*3 show the undigested PCR product sizes of 169 bp and 329 bp, respectively. The restriction patterns are indicative of the genotypes shown.
(Data kindly provided by K. Itoh, Department of Pharmaceutical Science, Akita
University Hospital, Hondo, Japan.)
Standard PCR
QIAGEN PCR and RT-PCR Application Guide
11

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M
The GRIN2B gene encodes a subunit of the glutamate
(N-methyl D-aspartate) receptor in the brain. This receptor subunit is expressed in the hippocampus and forebrain and is implicated in the processes of memory and learning.
GRIN2B may also be a candidate gene for the neurodegenerative disorder dentato-rubro-pallidoluysian atrophy
(DRPLA). This hereditary disease has symptoms similar to
Huntington’s disease, including epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Unlike Huntington’s, the onset for DRPLA can occur at any age. PCR analysis using
QIAGEN Taq DNA Polymerase distinguished two alleles of the GRIN2B gene for accurate genotyping ( Figure 9 ).
650 bp
Figure 9
Genomic DNA from 24 individuals was analyzed by PCR using the
QIAGEN Taq PCR Core Kit and primers specific for a fragment of the
GRIN2B gene. Homozygotes for allele type 1 yield a 650 bp amplicon, and homozygotes with type 2 yield a shorter PCR product.
Heterozygotes show both bands in PCR. M : markers.
(Data kindly provided by D.R. Muhleman, City of Hope, Duarte, CA,
USA.)
12 Standard PCR
QIAGEN PCR and RT-PCR Application Guide

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Because Taq DNA polymerase is active at ambient temperatures, extension of nonspecifically annealed primers and primer–dimers can occur during PCR setup and the initial heating step. These nonspecific PCR products compete with the amplification of specific products, reducing PCR yield and specificity. In hot-start PCR, the polymerase activity is prevented during reaction setup and starts only after the initial denaturation step. This can be performed manually by leaving out an essential PCR component (e.g., DNA polymerase, primers) and adding it only after the reaction mix heats up. For higher specificity and more convenience, an “automatic” hot start can be carried out using an inactivated DNA polymerase that is automatically activated by the initial heating step.
HotStarTaq DNA Polymerase is a chemically modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN developed for hot-start PCR. The combination of HotStarTaq
DNA Polymerase and the unique QIAGEN PCR Buffer minimizes nonspecific amplification products, primer–dimers, and background (see page 7). It is ideal for amplification reactions involving complex genomic or cDNA templates, very low-copy targets, or multiple primer pairs (see also
“Multiplex PCR”, page 23).
In this section, we present two examples of PCR amplification of pathogen genes and one example of amplification of the interleukin-6 gene using HotStarTaq DNA Polymerase.
E. coli of the serogroup O157 produce Vero cytotoxin, which has been responsible for a number of infectious outbreaks in recent years. People typically become infected through eating contaminated foods, particularly inadequately cooked ground beef or unpasteurized milk.
Accurate bacterial typing is important for epidemiological investigation of outbreaks. PCR analysis of the Vero cytotoxin gene using HotStarTaq DNA Polymerase, in combination with QIAGEN PCR Buffer, gave accurate and specific identification of the bacterial strain ( Figure 10 ).
Another Taq DNA polymerase and supplied buffer gave ambiguous results, due to low specificity in primer binding.
Figure 10
DNA from nine bacterial samples was analyzed by PCR using
HotStarTaq DNA Polymerase or Taq DNA polymerase from Supplier L following suppliers’ specifications. PCR was carried out for 35 cycles using primers for a 90 bp fragment of the Vero cytotoxin gene, specific for genotype VT2. PCR with HotStarTaq DNA Polymerase identified samples 3, 6, 7, and 9 as strains with the VT2 genotype. M : markers
(Data kindly provided by G. Smith, Central Public Health Laboratory,
London, UK.)
M 1 2 3 4
QIAGEN
5 6 7 8 9
M 1 2 3 4
Supplier L
5 6 7 8 9
90 bp
90 bp
Hot-start PCR
QIAGEN PCR and RT-PCR Application Guide
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Human herpes viruses are responsible for a number of clinically significant diseases. The herpes ICP6 gene, encoding the large subunit of ribonucleotide reductase, was cloned and used as a PCR template. Amplification was carried out using HotStarTaq DNA Polymerase.
Addition of Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA (see page 6), enabled amplification in this system, giving a strong, specific band in PCR ( Figure 11 ).
Figure 11
PCR of a cloned herpes ICP6 gene was carried out for 35 PCR cycles using HotStarTaq DNA Polymerase and primers specific for a 990 bp fragment of the gene. PCR was carried out with and without Q-Solution, as indicated. M : markers.
(Data kindly provided by K.M. Suling, Neuro-Oncology Department, Massachusetts General Hospital,
Charlestown, MA, USA.)
M
– +
Q-Solution
990 bp
M
QIAGEN Supplier P
II
Interleukin-6 (IL-6) is a cytokine involved in a large number of biological processes, including immunity, bone metabolism, reproduction, inflammation, neural development, and hematopoiesis. In immune responses, it induces B-cell and
T-cell differentiation, stimulates T-cell proliferation, and activates natural killer cells. Defects in IL-6 regulation contribute to a variety of disorders, such as osteoporosis, arthritis, autoimmune disorders, and tumor cell proliferation. Tumors stimulated by IL-6 include melanoma, leukemia, Kaposi’s sarcoma, and renal, prostatic, and ovarian carcinomas.
PCR of the IL-6 gene was carried out ( Figure 12 ). PCR using
HotStarTaq DNA Polymerase provided high specificity and low background without the need for optimization. PCR conditions were optimized for another hot-start DNA polymerase, but even this optimized system gave lower signals than did HotStarTaq DNA Polymerase.
320 bp
Figure 12
Genomic DNA was analyzed by PCR using HotStarTaq DNA
Polymerase or a hot-start DNA polymerase from Supplier P
II following suppliers’ specifications. PCR was carried out for 43 cycles using primers specific for an approximately 320 bp fragment of the IL-6 gene. M : 100 bp ladder.
(Data kindly provided by H. Trang, Toronto General Hospital,
Toronto, ON, Canada.)
14 Hot-start PCR
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PCR screening projects benefit greatly from reliable PCR methods that do not require extensive optimization. Robust amplification by QIAGEN Taq DNA Polymerase and
HotStarTaq DNA Polymerase provides efficient PCR for the wide range of differing templates screened without the need for optimization. In addition, using the Taq PCR Master Mix
Kit or the HotStarTaq Master Mix Kit simplifies both manual and automated PCR setup, resulting in significant time-saving with large numbers of reactions. Both Master Mix Kits contain premixed, ready-to-use solutions containing Taq or
HotStarTaq DNA Polymerase, PCR Buffer, MgCl
2
, and ultrapure dNTPs at optimized concentrations (see page 8).
Pipetting steps are minimized, reducing the possibility of errors and contamination.
In this section, we present several PCR screening applications for microarray construction, cDNA screening, DNA fingerprinting, screening for genetically modified organisms (GMOs), and amplification of multiple sequences from pre-amplified genomic DNA.
Primer-extension preamplification (PEP) is a method for whole-genome amplification starting with very small amounts of DNA, such as from a single cell. Multiple rounds of primer extension are carried out using a random mixture of oligonucleotides. This produces multiple copies of the DNA sequences originally present in the sample.
Although the method does not guarantee that all genomic sequences will be amplified, under the right conditions a large proportion of the genome can be amplified. After preamplification, PCR of specific gene sequences can then be carried out using only a small aliquot of sample.
The preamplified DNA can then be used as a template for highly specific amplification using HotStarTaq DNA
Polymerase ( Figure 13 ).
M M
Figure 13
120 PEP genomic DNA samples were analyzed by PCR for 45 amplification cycles using HotStarTaq DNA Polymerase. Fragment sizes ranging from 200 to 350 bp were analyzed. M : markers.
(Data kindly provided by J. Greenham, Incyte Europe Ltd.,
Cambridge, UK.)
PCR Screening
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cDNA libraries consist of many different cDNAs within the same vector sequences. Screening clones by PCR analysis using the Taq PCR Master Mix Kit simplified reaction setup and provided efficient amplification without the need for optimization ( Figure 14 ).
M
M
Figure 14
384 rat cDNA clones in a pT3T7 vector were screened by PCR using the QIAGEN Taq PCR Master Mix Kit. Inserts range from 0.5 kb to
2 kb. M : markers.
(Data kindly provided by Q. Guo and E. Liu, National Cancer
Institute, National Institutes of Health, Gaithersburg, MD, USA.)
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
PCR amplification is a crucial step in cDNA microarray construction. The thousands of cDNA species to be spotted on the array need to be amplified efficiently, accurately, and reproducibly in individual reactions. Using the
HotStarTaq Master Mix Kit simplifies the setup of these multiple reactions. HotStarTaq DNA Polymerase provided robust PCR performance for the different cDNA targets without the need for optimization ( Figure 15 ).
M 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
M 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Clone
Clone
Clone
Figure 15
48 cDNA inserts in a pUC18-derived plasmid were screened by PCR for 35 PCR cycles using the HotStarTaq Master Mix Kit and M13 primers. M : markers.
(Data kindly provided by I. Dzekunova, NIH/NCI Microarray Lab,
Gaithersburg, MD, USA.)
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M 1 2 3 4 5 6 7 8 9 10 11 12
Routine screening of plasmid inserts requires reliable amplification with minimal optimization. PCR of arabidopsis cDNA clones was simplified by use of the HotStarTaq
Master Mix Kit, allowing easy setup and reliable PCR amplification ( Figure 16 ).
Figure 16
Arabidopsis cDNA clones were screened by PCR for 30 cycles using the HotStarTaq Master Mix Kit and Q-Solution. PCR products were cleaned up using the QIAquick ® PCR Purification Kit. M : markers.
(Data kindly provided by S. Jyawook and J. Gill, NEN Life Science Products, Boston, MA, USA.)
Engineered herbicide resistance in crop plants provides an effective means of weed control. Potential outcrossing of these genetically modified organisms (GMOs), however, has become a significant environmental concern in recent years. In order to effectively control potential outcrossing, it is necessary to monitor how pollen, potentially containing engineered genes, is distributed. Using PCR analysis, pollen can be analyzed for presence of engineered genes, but this presents a challenge due to the limited amount of material. In addition, the pollen of interest may account for only a small percentage of the total pollen collected by bees. In this experiment, pollen collected by bees was analyzed by PCR for presence of the engineered pat herbicide-resistance gene. DNA purification using the
DNeasy ® Plant Mini Kit provided sufficient amounts of highquality DNA for PCR analysis. PCR using the Taq PCR Core
Kit gave a specific, strong band for all reactions ( Figure 17 ).
PCR with another Taq DNA polymerase gave much less efficient amplification.
M R P P
M R P P
QIAGEN
R P P R P P
Supplier X
R P P R P P
734 bp
398/400 bp
734 bp
398/400 bp
Figure 17
Pollen was collected from the pollen baskets of bees. DNA was purified using the DNeasy Mini Kit with herbicide-resistant rapeseed plants ( R ) and pollen derived from pollen baskets of bees ( P ). PCR was carried out using the Taq PCR Core Kit (QIAGEN) or another Taq DNA polymerase
(Supplier X) for 30 PCR cycles. Primers amplified a 734 bp fragment of the synthetic pat gene (left), and a second set of primers was used to amplify a 398 bp fragment of the same gene (middle). As a control, primers for a 400 bp fragment of a universal plant DNA sequence were used (right) as a confirmation. All samples tested showed presence of the pat gene. M : 100 bp marker.
(Data kindly provided by B. Hommel, R. Tober, and A.-G. Metke,
Federal Biological Research Center for Agriculture and Forestry (BBA),
Institute for Integrated Plant Protection, Kleinmachnow, Germany.)
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M +
QIAGEN
+ + – + +
Supplier S
II
+ – Template As genetically modified organisms (GMOs) become increasingly common and statutory regulations become more stringent, the need for accurate food-screening methods increases. Screening of foodstuffs and animal feed for presence of genetically modified material requires sensitive and accurate molecular techniques.
Using PCR analysis, soy products can be analyzed for presence of the CaMV 35S promoter, introduced into soybean plants by genetic engineering as the promoter for the EPSPS herbicide-resistance gene. PCR using
HotStarTaq DNA Polymerase and Q-Solution gave a specific, strong band for the 35S promoter. The Taq DNA polymerase from Supplier S
II gave high background and less specific PCR amplification ( Figure 18 ).
195 bp
Primers
Figure 18
DNA from soy lecithin powder was analyzed by PCR using HotStarTaq
DNA Polymerase and Q-Solution or Taq DNA polymerase from Supplier
S
II following suppliers’ specifications. PCR was carried out for 45 cycles using primers specific for a 195 bp fragment of the 35S CaMV promoter. M : markers.
(Data kindly provided by G. Mücher, GEN-IAL GmbH, Troisdorf,
Germany.)
QIAGEN CTAB
To test whether this screening method could be used for detection of GMO products in processed foods, PCR of the soy lectin gene, as a control, was carried out using
DNA from chocolate. Different DNA purification methods were tested. Since chocolate, like stool, is rich in PCR inhibitors, the QIAamp Stool Kit was used for DNA purification. Compared to a CTAB-precipitation method, DNA purification with the QIAamp Stool Kit gave a better template for PCR. PCR using HotStarTaq DNA Polymerase and Q-Solution gave efficient amplification and a strong band in PCR ( Figure 19 ).
150 bp
Primers
Figure 19
DNA was purified from chocolate using the QIAamp Stool Kit (QIAGEN) or a CTAB-precipitation method (CTAB), as indicated. PCR was carried out using HotStarTaq DNA Polymerase and Q-Solution for 45 cycles with primers specific for a 150 bp fragment of the soy lectin gene.
(Data kindly provided by G. Mücher, GEN-IAL GmbH, Troisdorf,
Germany.)
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Single-cell PCR provides a valuable tool for genetic characterization using a limited amount of starting material. By flow cytometry or micromanipulation, individual cells of interest can be isolated based on cell-surface markers or physical appearance. PCR from such a small sample requires a highly efficient PCR system (see “Single-cell PCR”, page 46). As with any experiment involving a single cell, there is no allowance for error: once the unique cell is used, it is not possible to repeat the experiment. The PCR must be extremely sensitive since the template is often only one or two gene copies. This limited amount of starting template can lead to formation of nonspecific PCR products such as primer–dimers. The stringent hot start provided by HotStarTaq DNA Polymerase, in combination with QIAGEN PCR Buffer, provides the highly sensitive and specific PCR required for single-cell analysis.
In this section, we present an example of single-cell PCR using
HotStarTaq DNA Polymerase.
For amplification of the single-copy p53 gene, the PCR template consists of only two copies. Three different hot-start
PCR systems were tested for amplification of this gene in murine cells. Individual cells were sorted, and amplification was carried out in parallel reactions, following guidelines for single-cell PCR (see page 46). Amplification with
HotStarTaq DNA Polymerase gave a strong specific signal.
In contrast, two other hot-start PCR systems gave large amounts of primer–dimers (Figure 20).
M H ot
St ar
Ta q
(Q
IA
G
EN
)
H ot
-s ta rt
e nz ym
(S up pl ie e r
P
)
II
A nt ib od ym ed ia te
(S up pl ie d r
L)
500 bp
Figure 20
A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes.
Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN ( HotStarTaq ), a hot-start enzyme and buffer from Supplier P
II
( Hot-start enzyme ), or antibody-mediated hot start and buffer from Supplier L ( round with 50 amplification cycles. M : markers.
Antibody-mediated ). PCR was carried out in one
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PCR templates with high GC-content or complex secondary structure are often difficult to amplify. A number of different additives, such as DMSO, formamide, and glycerol, can be added to PCR to modify the melting behavior of the DNA template and allow more efficient primer binding. Unlike these commonly used additives, Q-Solution, an innovative
PCR additive from QIAGEN, is used at just one working concentration, is nontoxic, and PCR purity is guaranteed (see page 6). Addition of Q-Solution often improves suboptimal
PCR in systems that do not work well under standard conditions, including amplification of GC-rich as well as other difficult templates. It can have varying effects, however, depending on the individual PCR assay. It is therefore often advisable to perform reactions in parallel with and without Q-Solution.
In this section, we present several examples of successful PCR using Q-Solution for GC-rich and other difficult templates that proved problematic with other PCR enzyme systems.
Arginosuccinate lyase catalyzes the last step in arginine biosynthesis. In the unicellular alga C. reinhardtii , this gene has a very high GC-content (>67%), making amplification especially difficult. Amplification of a 1.6 kb fragment using QIAGEN Taq DNA Polymerase and Q-Solution provided a strong, specific band, which was not possible with another Taq DNA polymerase ( Figure 21 ).
M +
QI
AG
EN
– C
Figure 21
PCR of a cDNA clone of the C. reinhardtii arginosuccinate lyase transcript was carried out for 45 PCR cycles using Taq DNA Polymerase from
QIAGEN or Supplier E
II following suppliers’ specifications. PCR with
QIAGEN Taq DNA Polymerase was carried out with and without
Q-Solution, as indicated. C : negative control with no template DNA.
M : markers
(Data kindly provided by F. Pfannenschmid and K. Haller, Institute for
Biochemistry, Genetics, and Microbiology, University of Regensburg,
Regensburg, Germany.)
Su pp lie r E
II
Q-Solution
1.6 kb
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Dystrophin is an important cytoskeletal protein found at the inner surface of muscle fibers. Deletions or duplications in the gene can lead to muscular dystrophy. Analysis of specific deletions provides information about the gene structure in individual cases. PCR of exons 21 and 34 of the dystrophin gene using QIAGEN Taq DNA Polymerase and Q-Solution gave highly specific results, allowing analysis of the gene structure in two muscular dystrophy patients. Another Taq DNA polymerase gave a large number of background bands ( Figure 22 ).
M
QIAGEN
34
A B C
21
A B C
34
A B C
Supplier X
21
A B C
178 bp
102 bp
Figure 22
DNA from two muscular dystrophy patients (B, C) and one unaffected individual (A) was analyzed by PCR using QIAGEN Taq DNA Polymerase and Q-Solution or Taq DNA Polymerase from Supplier X following suppliers’ specifications. PCR was carried out for 35 cycles using primers specific for a 178 bp fragment from exon 21 or a 102 bp fragment from exon 34 of the dystrophin gene. Patient C shows a deletion in both exon 21 and exon 34; patient B shows a deletion only in exon 21.
(Data kindly provided by H.-G. Katzera, Institute for Human Genetics, University of Hamburg, Hamburg, Germany.)
The tumor-supressor gene CDKN2A (cyclin-dependent kinase inhibitor 2A) is implicated in a number of different cancers, including familial malignant melanoma. PCR of exon 1 of this gene was carried out using HotStarTaq
DNA Polymerase compared with another Taq DNA polymerase. Q-Solution enabled amplification, giving a strong, specific band in PCR ( Figure 23 ).
M
QI
AG
EN
+ + – –
Su pp lie r X
Q-Solution
240 bp
Figure 23
DNA from human peripheral blood was analyzed by PCR using HotStarTaq DNA Polymerase and Q-Solution or Taq DNA Polymerase from Supplier X following suppliers’ specifications. PCR was carried out for 35 cycles using primers specific for a 240 bp fragment from exon 1 of the CDK2A gene. M : markers.
(Data kindly provided by J.-L. Blouin and G. Pelli, Medical Genetics, Cantonal Hospital and University of Geneva, Geneva, Switzerland.)
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PCR products of up to 3–4 kb can be routinely amplified using most standard PCR protocols. However, amplification of PCR products longer than 4 kb often fails without lengthy optimization. Reasons for failure are nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions, which have a greater effect on the amplification of longer PCR products than on shorter ones. longer fragments and allows for efficient amplification of fragments up to 7 kb (up to 10 kb with lambda DNA). In addition, Q-Solution, an innovative additive that modifies the melting behavior of nucleic acids, may help in some cases where secondary structure interferes with amplification of large fragments (see page 6). See pages 45 and 53 for guidelines to obtain longer PCR and RT-PCR products.
The uniquely balanced combination of cations in QIAGEN
PCR Buffer gives a high ratio of specific-to-nonspecific binding during the annealing step of every PCR cycle (see page 5).
This facilitates the highly specific binding required for PCR of
In this section, we present an example of amplification of large PCR products using QIAGEN Taq DNA Polymerase.
To test PCR efficiency with differently sized templates, PCR was carried out with primers for three differently sized fragments of human genomic DNA using two different Taq polymerases and buffers. PCR with QIAGEN Taq DNA
Polymerase and PCR Buffer gave highly specific and efficient amplification for all three PCR products. The Taq
DNA polymerase from Supplier P
II showed low PCR efficiency with the 1.5 kb fragment and, especially, the long 7.3 kb amplicon ( Figure 24 ).
M
Su pp lie r
P II
Q
IA
G
EN
Su pp lie r
P II
Q
IA
G
EN
Su pp lie r
P II
Q
IA
G
EN
– 7.3 kb
– 1.5 kb
– 0.5 kb
Figure 24
Three differently sized products from human genomic DNA were amplified using either QIAGEN Taq DNA Polymerase and PCR Buffer ( QIAGEN ) or Taq DNA polymerase and buffer from another supplier ( Supplier P
II
). Ten percent of each reaction was loaded on the gel. Results from duplicate
PCR amplifications are shown. M : markers.
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Multiplex PCR allows analysis of two or more targets simultaneously. This demanding PCR technique is used for genetic screening, microsatellite analysis, and other applications where it is necessary to amplify several products in a single reaction. This technique often requires extensive optimization because having multiple primer pairs in a single reaction increases the likelihood of primer–dimers and other nonspecific products that may interfere with the amplification of specific products. In addition, the concentrations of individual primer pairs often need to be optimized since different multiplex amplicons are often amplified with differing efficiencies, and multiple primer pairs can compete with each other in the reaction. (See page 47 for guidelines for multiplex PCR.)
In this section, we present several examples of successful multiplex PCR in identification of pathogens and analysis of dystrophin gene structure in muscular dystrophy (see also page 37 for multiplex RT-PCR applications).
M
P. intermedia
In inflammation of the gums, known as gingivitis, the total number of periodontal bacteria increases as well as the proportion of black-pigmented anaerobic rods (BPRs) among the total bacteria. This correlation implicates BPRs as a causative agent in gingivitis. Positive molecular identification of these BPRs is useful to analyze the microbiology of gingivitis. Multiplex PCR of 16S rRNA genes of different
BPRs allows detection of different species with the same reaction setup or within the same sample. In a pilot experiment, one master mix containing all three primer pairs was tested for PCR amplification of rRNA genes from three different bacterial species. While previous methods used in this laboratory gave a number of nonspecific bands, multiplex PCR with HotStarTaq DNA Polymerase provided highly specific PCR identification of the specific bacterial genes ( Figure 25 ).
P. nigrescens P. gingivalis
– 828 bp
– 404 bp
– 259 bp
Figure 25
Multiplex PCR of the indicated periodontal bacteria was carried out using HotStarTaq DNA Polymerase and primers specific for the 16S rRNA gene of Prevotella intermedia (259 bp), Prevotella nigrescens
(828 bp), and Porphyromonas gingivalis (404 bp). PCR was carried out for 35 PCR cycles with all primers in each reaction. M : 100 bp ladder.
(Data kindly provided by M. Okamato, Department of Oral
Bacteriology, Tsurumi University School of Dental Medicine,
Yokohama, Japan.)
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Dystrophin is an important cytoskeletal protein found at the inner surface of muscle fibers. This protein is reduced or missing in muscular dystrophy patients, usually corresponding to deletions and duplications in the dystrophin gene.
The gene is very complex, with 79 exons in humans and alternative splicing in different tissues. Multiplex PCR of the dystrophin gene provides an analysis of the gene structure and provides the potential to use dystrophin as a molecular marker for muscular dystrophy ( Figure 26 ).
Figure 26
Multiplex PCR was carried out using QIAGEN Taq DNA Polymerase for 30 PCR cycles with primers from, in order of fragment size, exons
52, 60, 47, 6, 13, 50, 43, 3, and the promoter region of the human dystrophin gene. Genomic DNA was analyzed from a normal and a muscular dystrophy patient. The muscular dystrophy patient was missing one of the PCR bands, possibly indicating a deletion in the gene. M : markers.
(Data kindly provided by A. Beckmann, E. Pascual, and J.M.
Schröder, Neuropathology Institute, University Hospital of the Rhine-
Westphalia Technical College, Aachen, Germany.)
M
No rm al
Pa tie nt
535 bp
410 bp
357 bp
271 bp
238 bp
202 bp
189 bp
139 bp
113 bp
M 1 2 3 4 5 6 7 8 9
P. carinii f. sp.
hominis pneumonia is common among HIVinfected individuals, transplant recipients, and other immunocompromised patients. In order to study P. carinii epidemiology, multiplex PCR of variable genomic regions with HotStarTaq DNA Polymerase was used to genotype specific strains of this pathogen ( Figure 27 ).
426 bp
340 bp
309 bp
204 bp
Figure 27
DNA was isolated from bronchoalveolar specimens of nine patients with P. carinii pneumonia using the QIAamp DNA Blood Mini Kit. Multiplex PCR was carried out using HotStarTaq DNA Polymerase and primers specific for the intron of the 26S rRNA gene (426 bp), the variable region of the mitochondrial 26S rRNA gene (340 bp), the region surrounding intron 6 of the β -tubulin gene (309 bp), and the internal transcribed spacer 1 of the nuclear rRNA gene operon (204 bp). All primers were used together in multiplex PCR for 40 cycles. M : markers.
(Data kindly provided by P. Hauser, Hospital Preventive Medicine, CHUV Hospital, Lausanne, Switzerland.)
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Adenoviruses are implicated in a number of different diseases, including conjunctivitis, sudden infant death syndrome (SIDS), and infantile viral gastroenteritis. The six different subgenera vary from the highly contagious and epidemic subgenus D adenoviruses to the relative harmless subgenus B and E adenoviruses. By sequencing the gene for the coat protein, subgenus-specific primers were designed and used in multiplex PCR with HotStarTaq DNA
Polymerase to distinguish adenoviral subgenera ( Figure 28A ).
DNA from clinical swab and stool samples can be difficult to use in PCR due to small sample amounts in swab samples and high levels of PCR inhibitors in stool samples.
DNA purification using the QIAamp ® DNA Blood Mini Kit provided high-quality DNA for PCR from both sample types ( Figure 28B ). (Note: The QIAamp Stool Kit is now available for purification of DNA from stool samples.)
A
M
B
M S
+
S
A+
C
B+
D
E+
D
S T O O
B+
E+
D
A+
B+
E+
D
Subgenus
F (653 bp)
B (517 bp)
E (453 bp)
D (394 bp)
A (298 bp)
C (234 bp)
F (653 bp)
B (517 bp)
E (453 bp)
D (394 bp)
C (234 bp)
Figure 28
■
Mixtures of two, three, or four different purified subgenus-specific adenoviral DNA standards, as indicated, were used as templates in multiplex PCR. PCR was carried out using HotStar Taq DNA Polymerase and primers specific for adenovirus subgenus A,B,C,D,E, and F. All primers were used together in multiplex PCR for 40 PCR cycles. + : As a DNA size ladder, PCR products from six individual subgenus-specific reactions were mixed together before loading the gel. M : markers.
■
Total DNA was isolated from clinical stool ( S ), ocular swab ( O ), and throat swab ( T ) samples using the QIAamp DNA Blood Mini Kit. Multiplex
PCR was carried out using HotStarTaq DNA Polymerase and primers specific for adenovirus subgenus A, B, C, D, E, and F. All primers were used together in multiplex PCR for 40 PCR cycles. Subgenus-specific bands are indicated. M : markers.
(Data kindly provided by P. Pring-Åkerblom, Institute for Virology, Medical School of Hanover, Hanover, Germany. Data from Pring-Åkerbloom, P.,
Trijsenaar, F.E.J., Adrian, T., and Hoyer, H. [1999] Multiplex polymerase chain reaction for subgenus-specific detection of human adenoviruses in clinical samples. J. Med. Virol. 58 , 87. © 1999 Wiley-Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)
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Tissue samples are often preserved by embedding in paraffin.
Archives of preserved tissues provide a vast resource for retrospective studies and provide the opportunity to analyze disease progress over a long period of time. In some cases, as with rare diseases, paraffin-embedded samples may be the only available representatives of disease tissue. tissue with formalin, prior to paraffin embedding, cross-links the DNA to protein in the sample, making the DNA rigid and ill-suited for PCR, even with shorter fragments. A robust PCR system is necessary for efficient amplification of this sort of difficult template. The QIAGEN Taq DNA Polymerase and the innovative QIAGEN PCR Buffer facilitate PCR over a wide range of PCR conditions without optimization (see page 5).
This robust combination makes challenging PCR possible, such as with DNA from paraffin-embedded tissues.
While paraffin embedding aids later microscopic and histological analyses, PCR of DNA from paraffin-embedded tissues can be difficult. The paraffin can interfere with DNA isolation, and the fixation and paraffin-embedding processes inevitably cause some DNA degradation. Since DNA from paraffin-embedded tissues is typically <650 bp, PCR of larger fragments is generally not possible. In addition, fixation of the
In this section, we present three examples of PCR with genomic
DNA from paraffin-embedded tissues using QIAGEN Taq
DNA Polymerase.
Exon 1
M
Von Hippel–Lindau (VHL) disease is a genetic disorder leading to blood-vessel tumors (hemangiomas), which can occur in many places throughout the body. The disease is the result of a variety of differing mutations in the VHL gene.
Successful DNA isolation with the QIAamp DNA Mini Kit gave high-quality DNA for PCR analysis using QIAGEN
Taq DNA Polymerase. PCR of specific exons of the VHL gene, followed by sequencing, was used to analyze the gene structure in paraffin-embedded tissue samples from four renal cell carcinoma patients ( Figure 29 ).
M
Exon 2
M
Exon 3
Figure 29
DNA was isolated from paraffin-embedded tissues of four renal cell carcinoma patients using the QIAamp DNA Mini Kit. PCR was carried out for 35 PCR cycles using QIAGEN Taq DNA Polymerase and primers specific for fragments of the indicated exons of the VHL gene.
M : markers.
(Data kindly provided by P. Schraml, Institute for Pathology, Cantonal
Hospital and University of Basel, Basel, Switzerland.)
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M —
Sample 1
H B
Identification of mycobacterial infection is important for proper clinical diagnosis and treatment. In some cases, where the infection was not suspected and no fresh clinical specimens taken for culture, paraffin-embedded samples must be used. PCR methods using QIAGEN Taq DNA
Polymerase, followed by restriction digest, allowed sensitive detection and determination of Mycobacterium species in paraffin-embedded tissues ( Figure 30 ).
—
Sample 2
H B
142 bp
Figure 30
PCR was carried out for 35 PCR cycles using the QIAGEN Taq PCR Core Kit and primers specific for a 142 bp fragment of the Mycobacterium
65 kDa antigen gene. Following PCR, the reaction was cleaned up using the QIAquick PCR Purification Kit and digested with Hha I ( H ) or Bst UI ( B ) restriction enzymes, as indicated. The restriction pattern for both samples is indicative of the species M. tuberculosis .
(Data kindly provided by M. Watanabe, Second Department of Pathology, Mie University School of Medicine, Tsu, Japan.)
M G7
Sample 1
C7 G8 A8 G7
Sample 2
C7 G8 A8
Cerebrotendinous xanthomatosis (CTX) is a hereditary lipid-storage disease caused by mutations in the cytochrome P450
27 gene ( CYP27 ). The disease is characterized by yellowish fatty tumors on the tendons, most often on the Achilles tendon. Patients with CTX may start with mild mental retardation and then develop seizures, emotional or psychiatric disturbances, and motor deficits. Point mutations in exon 7 and exon 8 of the CYP27 gene can be identified by PCR using allele-specific primers that contain these point mutations. Specific point mutations were detected in two paraffin-embedded samples analyzed by PCR using
QIAGEN Taq DNA Polymerase ( Figure 31 ).
Figure 31
PCR was carried out for 35 PCR cycles using the QIAGEN Taq PCR Core Kit with Q-Solution and primers specific for a fragment of exon 7 or exon 8 of the CYP27 gene. Primers were specific for either the wild-type sequences (C7 and G8), a C to G point mutation in exon 7 (G7), or a
G to A mutation in exon 8 (A8). Both samples shown here were wild-type for exon 8. Sample 2 was wild-type for exon 7 whereas, for sample 1, the results suggest another form of mutation in exon 7. M : markers.
(Data kindly provided by M. Watanabe, Second Department of Pathology, Mie University School of Medicine, Tsu, Japan.)
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Methylation-specific PCR (MSP) allows precise mapping of
DNA methylation patterns in GC-rich regions of DNA (see box below for details about the MSP technique).
Hypermethylation of promoter regions is often a decisive factor in inactivation of tumor suppressor genes in human cancers. Since methylation patterns are tumor-specific, mapping them has become an important tool for understanding gene expression in tumors.
In this section, we present two methylation mapping studies using HotStarTaq DNA Polymerase in MSP.
In higher-order eukaryotes, DNA is methylated only at certain cytosines located 5' to a guanosine. This occurs especially in GC-rich regions, known as CpG islands. To distinguish the methylation state of a sequence, MSP relies on differential chemical modification of cytosine residues in DNA. Treatment with sodium bisulfite converts unmethylated cytosine residues into uracil, leaving the methylated cytosines unchanged. This modification thus creates different DNA sequences for methylated and unmethylated DNA. PCR primers can than be designed so as to distinguish between these different sequences.
Two sets of primers are designed: one set with sequences containing unchanged (methylated in the genomic DNA) cytosines and the other set with sequences containing the altered (unmethylated in the genomic DNA) cytosines. A comparison of PCR results using the two sets of primers reveals the methylation state of the DNA. If the primer set with the altered sequence gives a PCR product, then the indicated cytosine was unmethylated. If the primer set with the unchanged sequence gives a PCR product, then the cytosines were methylated and thus protected from alteration.
B C C
Hypermethylation of the promoter of the glutathione
S-transferase gene P1 ( GSTP1 ) is the most frequent DNA alteration in protastic carcinomas. Hypermethylation is present in >90% of prostatic carcinomas and absent in normal or benign hyperplastic prostatic tissue. This provides an excellent molecular marker for prostate cancer. While traditional methods for MSP use high concentrations of
Mg 2+ , dNTPs, and primers, the HotStarTaq Master Mix Kit provided simple and effective MSP under standard conditions without optimizing ( Figure 32 ).
99 bp
92 bp
Figure 32
DNA was isolated from prostatic carcinoma ( C ) and benign hyperplastic prostatic ( B ) tissue using the QIAamp DNA Mini Kit. DNA was modified with bisulfite and MSP was carried out using the HotStarTaq DNA Polymerase Master Mix Kit for 32 PCR cycles. Primers were specific for a
92 bp fragment of the GSTP1 gene, derived from methylated sequence (blue), or a 99 bp fragment derived from unmethylated sequence (green).
PCR products were resolved on a polyacrylamide gel and visualized using laser-fluorescence detection. The carcinoma sample shows presence of both methylated and unmethylated sequences due to presence of normal cells in the sample.
(Data kindly provided by C. Goessl, Urological Clinic and Polyclinic, Benjamin Franklin Hospital of the Free University Berlin, Germany.)
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Hypermethylation of the promoter region is implicated in regulation of tumor-supressor genes CDKN2A and
CDKN2B in malignant gliomas (tumors of glial cells in the brain). Differential methylation patterns can be detected using MSP. HotStarTaq DNA Polymerase was compared with another Taq DNA polymerase for performance in MSP
( Figure 33 ).
QIAGEN
CDKN2A
0 M 0
CDKN2B
0
CDKN2A
Supplier S
M 0
CDKN2B
Figure 33
DNA was modified with bisulfite and MSP was carried out using
HotStarTaq DNA Polymerase or Taq DNA Polymerase from Supplier S for 40 PCR cycles. Primers were specific for a 254 bp fragment of the
CDKN2A gene or a 720 bp fragment of the CDKN2B gene. 0 : negative control (no template); M : markers.
(Data kindly supplied by M. Walter, Neuropathology Institute,
Heinrich Heine University, Düsseldorf, Germany.)
720 bp
254 bp
720 bp
254 bp
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Random-amplified polymorphic DNA PCR (RAPD-PCR) is often used for genomic characterization of plant species, which generally have fewer well-characterized molecular markers than animal species (see box for details). Generally,
RAPD-PCR is performed under conditions of low stringency to allow priming where the sequence does not exactly match the genomic DNA. Highly reproducible PCR results under low stringency conditions are needed to obtain useful RAPD-PCR fingerprints. The QIAGEN Taq DNA Polymerase and the innovative QIAGEN PCR Buffer facilitate PCR over a wide range of PCR conditions without optimization (see page 5).
This robust combination provides reliable and reproducible results for RAPD-PCR.
In this section, we present an example of RAPD-PCR fingerprinting using QIAGEN Taq DNA Polymerase.
RAPD-PCR makes use of random oligomers under low-stringency priming conditions to prime PCR from multiple sites in genomic DNA where the primers fortuitously match or almost match. PCR is carried out using one or more defined random oligomers. Each PCR results in a set of amplified fragments corresponding to specific, but unknown, fragments of genomic
DNA where the primers bind. This provides a distinct amplification pattern that can be used for DNA fingerprinting. Singlebase variations in the DNA where the primer binds or deletions/insertions between the primer binding sites give an altered
PCR pattern and therefore a different DNA fingerprint. Resolving the fragments on a gel allows side-by-side comparison of different individual genotypes.
Parents F
1 generation
M ➁ ❹ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
RAPD-PCR was used for fingerprint analysis of the F
1 generation from two haploid potato plants. QIAGEN Taq DNA Polymerase provided specific PCR amplification so that the F
1 generation plants could be analyzed
( Figure 34 ).
Figure 34
Genomic DNA from potato callus tissue was analyzed by RAPD-PCR for 40 PCR cycles using QIAGEN Taq DNA Polymerase and random
12mer primers. M : markers.
(Data kindly provided by A. Loessl, Department of Horticulture and Plant Breeding, University of Technology Munich, Munich, Germany.)
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In two-step RT-PCR, reverse transcription is carried out in one reaction, and an aliquot of the RT reaction is used in a separate PCR. This has the advantage that one RT reaction can be used for multiple PCR, and both steps can be optimized separately if necessary.
Successful RT-PCR depends on the efficiency of both the RT and the PCR steps. Low yields and nonspecific priming in the
RT step result in decreased sensitivity of the entire RT-PCR process. Omniscript Reverse Transcriptase has a high affinity for RNA to provide highly specific and sensitive reverse transcription (see page 9).
susceptible to nonspecific priming in the PCR step due to their single-stranded nature. The uniquely balanced combination of cations in QIAGEN PCR Buffer allows a high ratio of specific-to-nonspecific binding during the annealing step of every PCR cycle (see page 5). This facilitates the highly specific binding required for PCR of single-stranded cDNAs in two-step RT-PCR. When even higher specificity is needed, a hot start for the PCR, using HotStarTaq DNA Polymerase (see page 7), can eliminate nonspecific amplification products, ensuring highly specific and reproducible two-step RT-PCR.
Compared to double-stranded DNA templates used in PCR, cDNAs produced during reverse transcription are more
In this section, we present several two-step RT-PCR applications using Omniscript Reverse Transcriptase, QIAGEN Taq DNA
Polymerase, and HotStarTaq DNA Polymerase for viral detection and analysis of gene expression.
M – + RT
Interleukin-10 is an anti-inflammatory cytokine and a key regulatory component of the immune response and other pathological processes. It suppresses pre-inflammatory cytokines and stimulates MHC II expression, immunoglobin secretion, proliferating B-cells, and T-cells. This cytokine also has an important role in costimulation of thymocytes and in mast-cell growth. The anti-inflammatory action of this protein may have clinical benefits for arthritis and other conditions involving inflammation. RT-PCR of the rat interleukin-10 transcript was carried out ( Figure 35 ). HotStarTaq
DNA Polymerase in the PCR step provided high specificity and low background in two-step RT-PCR.
680 bp
Figure 35
Following reverse transcription of rat RNA, PCR was carried out for
30 PCR cycles using HotStarTaq DNA Polymerase and primers specific for a 680 bp fragment of the interleukin-10 transcript. M : markers.
(Data kindly provided by H. Kitasato, Kitasato University School of
Medicine, Sagamihara-shi, Japan.)
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®
Quantitative, real-time RT-PCR is possible using TaqMan technology or other systems such as the LightCycler ® and iCycler ™ . Quantitation is based on the threshold cycle
(C
T
), the first PCR cycle with a detectable fluorescent signal in relation to an internal standard. A lower C
T value indicates
TNF-
40 detection at an earlier cycle and thus a larger amount of the original mRNA. Reverse transcription with Omniscript
RT gave detection 5 to 8 cycles earlier than when using another RT ( Figure 36 ). This corresponds to an increased sensitivity of >30-fold.
-Actin
30
35
25
30
25
20
15
5
0
MM
LV
RN as
- RT e H
Om nis cri pt
RT
0
MM
LV
RN as
- RT e H
Om nis cri pt
RT
Figure 36
Real-time RT-PCR analysis of TNFα mRNA and, as a control, β -actin mRNA was performed using Omniscript (QIAGEN) or an MMLV RNase H–
(Supplier L) reverse transcriptase in two-step RT-PCR. The graph shows the number of PCR cycles needed to detect the amplicon (threshold cycle,
C
T
), a highly sensitive measure of relative template concentration.
(Data kindly provided by S. Martin, German Diabetes Research Institute, Düsseldorf, Germany.)
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Nested RT-PCR can be useful for highly sensitive detection of low-abundance transcripts or using small amounts of template. Following reverse transcription, nested PCR is carried out with two rounds of amplification reactions. The second-round PCR is performed with two primers that hybridize to sequences internal to the first-round primer–target sequences so that only specific first-round PCR products will be amplified in the second round (see page 48 for more details and guidelines for nested PCR). This technique was used for highly sensitive detection of human myosin heavy chain mRNA using HotStarTaq DNA Polymerase
( Figure 37 ).
M + – RT
424 bp
Figure 37
Following reverse transcription, nested PCR was carried out in two rounds of 35 and 20 cycles, respectively. HotStarTaq DNA
Polymerase was used with Q-Solution in the PCR to amplify a
424 bp fragment of the human myosin heavy chain mRNA.
M : markers.
(Data kindly provided by H. Sato, Legal Medicine, Yokohama
City University, Yokohama, Japan.)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-characterized enzyme with a key role in glycolysis.
GAPDH is a so-called “housekeeping” gene, whose expression remains constant under a wide variety of physiological conditions. For this reason, GADPH expression is commonly used as a standard in quantitative and semiquantitative
RT-PCR. Two-step RT-PCR of the GAPDH transcript in rat and mouse liver was carried out ( Figure 38 ). The combination of the high-affinity Omniscript Reverse Transcriptase in the
RT step and the high specificity of HotStarTaq DNA
Polymerase in the PCR step provide low background and a single band in RT-PCR.
Mouse Mouse
M + – + – +
Rat
– RT
462 bp
Figure 38
Total RNA was purified from rat and mouse liver using the RNeasy
Mini Kit. Reverse transcription was carried out using the Omniscript RT
Kit. PCR was carried out for 35 PCR cycles using HotStarTaq DNA
Polymerase and primers specific for a 462 bp fragment of the GAPDH transcript. M : markers.
(Data kindly provided by T. Sasahara, Kitasato University School of
Medicine, Sagamihara-shi, Japan.)
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M 2 1 0.5
0.2
µg RNA
In eukaryotes, pre-mRNAs are processed in the nucleus with addition of a 5' cap and poly-A tail and splicing out introns. Studying nuclear RNA provides important information about processing, regulation, and transport of RNA species. Obtaining nuclear RNA can be difficult, especially in plants. The nuclei must first be separated from the other more abundant cellular organelles, such as chloroplasts and mitochondria, and from the major store of RNA in the cytosol. The nuclei themselves contain large amounts of
DNA and only relatively small amounts of nuclear RNA.
RT-PCR from such small amounts of starting material can be difficult. With QIAGEN Taq DNA Polymerase, two-step
RT-PCR was used to efficiently amplify nuclear RNA species
( Figure 39 ).
Figure 39
Two-step RT-PCR was carried out using the indicated amounts of nuclear RNA from tobacco leaves. The PCR step was carried out for up to 30 PCR cycles using QIAGEN Taq DNA Polymerase and primers specific for β -actin. M : markers.
(Data kindly provided by H. Kodama, Department of Bioresources
Chemistry, Chiba University, Matsudo, Japan.)
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M β β β
Barley stripe mosaic virus (BSMV) is an RNA virus infecting barley, wheat, and several grass species. Infection with
BSMV leads to chlorotic mottling with yellow stripes or spots. This virus is unique for its very high level of seed transmission. BSMV infections, which have been reported worldwide, result in a significant reduction in agricultural yield of barley and wheat along with a lengthened time to harvest. Barley and wheat seeds are routinely checked for presence of BSMV, which can now be done by PCR analysis.
Omniscript Reverse Transcriptase and QIAGEN Taq DNA
Polymerase gave successful RT-PCR amplification and detection of BSMV RNA (Figure 40 ).
400 bp
Figure 40
Total RNA was isolated using the RNeasy Plant Mini Kit. RNA was reverse transcribed using the Omniscript RT Kit and oligo-dT primer. PCR was carried out for 35 cycles using the QIAGEN Taq PCR Core Kit with Q-Solution and primers specific for a 400 bp fragment of the BSMV RNA β .
M : markers.
(Data kindly provided by A.D. Stewart, Department of Biology, Emory University, Atlanta, GA, USA.)
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In one-step RT-PCR, all primers and components are added in one tube, using gene-specific primers that serve in both reverse transcription and PCR. During reverse transcription, the temperature needs to be high enough for specific primer annealing and resolution of RNA secondary structure but low enough that Taq DNA polymerase is mostly inactive. After allowing sufficient time for reverse transcription, the temperature is raised for PCR. This inactivates the thermolabile reverse transcriptase and allows the high-temperature DNA polymerase to amplify the cDNA.
oped reaction buffer. The QIAGEN OneStep RT-PCR Buffer* is designed to enable both efficient reverse transcription and specific amplification (see page 10). As with all QIAGEN
PCR buffers, the QIAGEN OneStep RT-PCR Buffer provides the advantage of a balanced combination of cations to ensure specific primer annealing with minimal optimization.
Since both the reverse-transcription reaction and PCR are performed in the same buffer, it is not possible to separately optimize the two reactions. The reverse transcription step can have poor efficiency with traditional PCR buffers. In addition, any DNA polymerase activity during reverse transcription can lead to nonspecific priming and low sensitivity, especially since the cDNAs produced during reverse transcription are highly susceptible to nonspecific priming due to their singlestranded nature.
During reverse transcription, HotStarTaq DNA Polymerase
(see page 7) in the enzyme mix is completely inactive and does not interfere with the RT reaction or nonspecifically prime PCR at the lower RT temperature. A simple heating step simultaneously switches on the polymerase activity and switches off reverse transcripition, inactivating the reverse transcriptases. This ensures temporal separation of reverse transcription and PCR, allowing both processes to be performed sequentially in one tube.
In this section, we present several one-step RT-PCR applications using the QIAGEN OneStep RT-PCR Kit for viral detection, gene-expression analysis, and multiplex one-step RT-PCR.
The QIAGEN OneStep RT-PCR Kit solves these problems by means of a unique enzyme combination and specially devel-
* Patent pending
36
M 1 2 3 4
Simian immunodeficiency virus (SIV) is a retrovirus infecting
African monkeys. SIV is closely related to HIV and is believed to be a progenitor species. The QIAGEN
OneStep RT-PCR Kit was used to detect expression of the
SIV gp130 gene, encoding an envelope glycoprotein, in sooty mangabey, an African primate ( Figure 41 ). The appearance of a single strong band demonstrates the high specificity and sensitivity of the QIAGEN OneStep RT-PCR Kit.
500 bp
Figure 41
RT-PCR was carried out using the QIAGEN OneStep RT-PCR Kit. A 500 bp fragment of the SIV gp130 gene was amplified in 40 PCR cycles. M : markers.
(Data kindly provided by L. Perry, Yerkes Vaccine Research Center, Emory University, Atlanta, GA, USA.)
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Collagenase is a matrix metalloproteinase that is overexpressed in a number of inflammatory states. Collagenase induction by the phorbol ester PMA (phorbol 12-myristate
13-acetate) was studied in rheumatoid arthritis synovial fibroblasts. RNA was isolated using the RNeasy Mini Kit and used directly in one-step RT-PCR with the QIAGEN
OneStep RT-PCR Kit ( Figure 42 ).
M
721 bp
274 bp
Figure 42
Rheumatoid arthritis synovial fibroblasts were induced overnight with PMA. RNA was isolated using the RNeasy Mini Kit. The RNA was used directly in RT-PCR with the QIAGEN OneStep RT-PCR Kit using primers for a 721 bp fragment of the collagenase-1 gene or, as a control, a 274 bp fragment of the cyclophilin gene for 30 PCR cycles. The double band for cyclophilin correlates to two alternatively spliced transcripts. M : markers.
(Data kindly provided by B. Moore, Department of Biochemistry, University of Alabama, Birmingham, AL, USA.)
Multiplex RT-PCR allows analysis of two or more targets simultaneously. Studying expression of wnt-1 , a protooncogene and key regulator of animal development, a fragment of the β -actin gene was simultaneously amplified as an internal control ( Figure 43 ). A common difficulty with multiplex RT-PCR is that transcripts with different abundance are amplified in the same reaction (see also
“Multiplex PCR”, page 23). Since β -actin is much more abundant than the wnt-1 transcript, the signal from β -actin is much more prevalent. With the QIAGEN OneStep
RT-PCR Kit, both transcripts were efficiently amplified in a single reaction without optimization.
M — +
636 bp
215 bp
Figure 43
Multiplex RT-PCR was carried out using the QIAGEN OneStep RT-PCR Kit and primers for a 636 bp fragment of the wnt-1 gene and a 215 bp fragment of the β -actin gene. PCR was carried out for 25 cycles. – : negative control ( β -actin only); M : markers.
(Data kindly provided by O. Huber and T. Hug, Institute of Clinical Chemistry and Pathobiochemistry, Benjamin Franklin University Hospital of the
Free University Berlin, Germany.)
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Single-cell RT-PCR provides a valuable tool for gene expression analysis of individual cells of interest. By flow cytometry or micromanipulation, single cells can be isolated based on cellsurface markers or physical appearance. PCR from such a small sample requires highly efficient RT-PCR system (see also
“Single-Cell PCR”, page 19).
ground smear. Sensiscript Reverse Transcriptase, one of the two high-affinity reverse transcriptases in the QIAGEN
OneStep Enzyme Mix, is specially developed for highly sensitive applications with very small amounts of RNA (see page 9), giving excellent performance in single-cell RT-PCR. A simple heating step simultaneously activates HotStarTaq DNA
Polymerase in the enzyme mix and inactivates the reverse transcriptases (see page 7) to ensure highly specific and reproducible RT-PCR even with complex systems.
In single-cell RT-PCR, not only the PCR but also the RT sensitivity is critical. In a one-step RT-PCR system, single-cell RT-
PCR proves a particular challenge, since both RT and PCR take place in the same tube, the system must be designed so as to maximize both RT and PCR efficiency. It is especially critical that the DNA polymerase remain inactive during reverse transcription. This helps to eliminate any nonspecific amplification products, such as primer–dimers and back-
In this section, we present two single-cell RT-PCR applications using the QIAGEN OneStep RT-PCR Kit and QIAGEN Taq DNA
Polymerase for analysis of sorted B-cells and individual neurons.
Single murine B-cell precursors were isolated from bone marrow, sorted by flow cytometry, and analyzed for the presence or absence of expression of kappa light-chain gene rearrangements. The copy number of this low-copy transcript is currently unknown and depends on the developmental stage of the cell. In five of the ten cells tested, no expression of the gene was detected; the other five cells showed a clear signal ( Figure 44A ). All ten cells gave a strong signal for β -actin ( Figure 44B ), indicating that the
QIAGEN OneStep RT-PCR system worked successfully with each individual cell.
A M 1 2 3 4 5 6 7 8 9 10
B M 1 2 3 4 5 6 7 8 9 10
350 bp
550 bp
Figure 44
An approximately 350 nt fragment of the kappa light-chain transcript and a 550 nt fragment of the β -actin transcript were amplified from single cells ( 1–10 ) in multiplex RT-PCR using the QIAGEN OneStep RT-PCR Kit. Following multiplex RT-PCR, the reaction was split into two aliquots, and nested primers were used in PCR with QIAGEN Taq DNA Polymerase to amplify either
■ the fragment of the β -actin transcript. M : markers.
■ t he fragment of the kappa light-chain transcript or
(Data kindly provided by S. Schwers and A. Ehlich, University of Cologne, Germany.)
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A M α 2 α 3 α 4 α 5 α 6 α 7 β 2 β 3 β 4 Subunit
Nicotinic acetylcholine receptors (nACHRs) in the brain are ligand-gated ion channels assembled from up to nine different subunits. Expression of different subunits in individual neurons was correlated with electrophysiological responses to nicotine. Two distinct neuron types were characterized.
Type A neurons expressed the α 3, α 4, β 2, and β 4 nACHR subunits ( Figure 45A ) and showed a larger electrophysiological response to nicotine than to cytisine, a nicotinic agonist. Type B neurons gave a larger response to cytisine than to nicotine. These neurons showed expression of the
α 6, β 2, and β 3 nACHR subunits ( Figure 45B ). Sensitive and specific PCR of individual neurons using QIAGEN Taq
DNA Polymerase clearly distinguished these two cell types.
B M α 2 α 3 α 4 α 5 α 6 α 7 β 2 β 3 β 4 Subunit
Figure 45
The cytoplasm from individual locus ceruleus neurons of rats was aspirated with drawn glass pipets. Multiplex RT-PCR was carried out using
QIAGEN Taq DNA Polymerase in the PCR step for 20 cycles, with primers for all the nACHR subunits. Aliquots with 1/50 of this PCR were used in separate PCRs for 40 cycles using only the primer pair specific to one of the subunits. A portion of these reactions was purified using the QIAquick
PCR Purification Kit and used for restriction analysis to confirm the identity of the PCR product (not shown). M : markers.
(Data kindly provided by A. de Kerchove d’Exaerde, Molecular Neurobiology, Pasteur Institute, Paris, France. Data from Léna, C. et al [1999]
Proc. Natl. Acad. Sci. USA 96 , 12126. Published with permission of The National Academy of Sciences USA. This must not imply endorsement by The National Academies or the Proceedings of the National Academy of Sciences [PNAS].)
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Differential display (DD) is a powerful molecular technique for studying differential gene expression (see box for details). The simplicity, sensitivity, and reproducibility of DD make it suitable for use in a number of fields. With DD, it is also relatively simple to clone and sequence differentially expressed products, which can be directly excised form the DD gel.
cDNAs produced in the reverse-transcription step of DD are more susceptible to nonspecific priming in the PCR step due to their single-stranded nature. The uniquely balanced combination of cations in QIAGEN PCR Buffer (see page 5) allows a high ratio of specific-to-nonspecific binding during the annealing step of every PCR cycle in DD. This facilitates the highly specific binding required for the PCR step of DD.
As with other RT-PCR applications, successful DD depends on the efficiency of both the RT and the PCR steps. Low yields and nonspecific priming in the RT step result in decreased sensitivity of the entire DD process. Sensiscript Reverse
Transcriptase provides highly sensitive reverse transcription with small amounts of RNA (see page 9) for efficient DD with limited starting material.
In this section, we present two examples of successful DD using Sensiscript Reverse Transcriptase and QIAGEN Taq
DNA Polymerase for analyses of gene expression in pancreatitis and human prostate cancer.
In some cases, RNA amounts for DD are limiting, reducing sensitivity. This is particularly the case when studying gene expression of rare populations of cells or where the cells of interest represent a small percentage of the total. Higher sensitivity in DD is needed in these cases. QIAGEN
Sensiscript RT was compared to the MMLV RNase H – RT from Supplier L for performance in DD. With Sensiscript RT, longer DD products were detected that were lost when using the other RT. The sensitivity was considerably higher so that it was possible to use 100-fold less RNA per DD reaction ( Figure 46 ).
0
Supplier L
(MMLV RNase H-)
4 16 80
QIAGEN
(Sensiscript)
20
0
0 4 16 80 20
0 ng RNA
— 300 bp
Figure 46
DD was performed with the indicated amounts of total RNA from LNCaP (human prostate cancer) cells using the RNAimage
The RT step was carried out with an oligo-dT anchor primer using MMLV RNase H –
® Kit (GenHunter Corp.).
(Supplier L) or Sensiscript RT (QIAGEN). Amplification was carried out using arbitrary 13mer primers and 33 P-dATP to allow visualization by autoradiography.
(Data kindly provided by I. Bosch, H. Melichar, and A.B. Pardee, Division of Cancer Biology, Dana-Farber Cancer Institute and Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. Data from Bosch, I. et al [2000] Nucl. Acids Res.
28 , e27. Published with permission of Oxford University Press.)
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Differential display (DD) is a multiplex RT-PCR technique to analyze differential gene expression. The process begins with reverse transcription using one of three primers that anchor to the poly-A tails at the 3' ends of mRNAs (i.e., T n
G, T n
C, or
T n
A, which define the three possible junctions of the poly-A tail). Multiplex PCR is then carried out using a defined arbitrary oligomer as the upstream primer. Each RT-PCR results in a set of amplified fragments corresponding to the 3' ends of various mRNAs. Resolving the fragments on a denaturing polyacrylamide gel gives a “fingerprint” of gene expression and allows side-by-side comparison of different mRNA populations. Differentially displayed fragments can then be excised from the gel for sequencing and cloning.
DD was used to identify genes that may play a role in chronic pancreatitis (severe inflammation of the pancreas).
Gene expression was investigated in rats with experimentally induced pancreatitis and compared to normal gene expression in control rats. Compared to another Taq DNA polymerase, DD using QIAGEN Taq DNA Polymerase provided a more consistent pattern of amplification products over a wider range of product sizes ( Figure 47 ).
G c12
QIAGEN
C c12
A c12
G c12
Supplier P
II
C c12
A c12
Figure 47
Differential display reactions using QIAGEN Taq DNA Polymerase and
QIAGEN PCR Buffer ( QIAGEN ) and Taq DNA polymerase and PCR buffer from another supplier ( Supplier P
II
). Results obtained using three different oligo(dT
11
) primers bearing an A, C, or G at the 3’ position
( A , C , G ) are displayed. c : healthy control animal; 1 , 2 : animals suffering from induced pancreatitis resulting from treatment with different amounts of the same chemical substance. Arrows indicate differences with respect to the presence/absence or strength of the signal between samples from experimental animals and the relevant control.
(Data kindly provided by A. Wilmen and P. Füller, Institute for Internal medicine, Philipps University, Marburg, Germany.)
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Quantitative analysis of gene expression for entire mRNA populations is practical using SAGE ™ technology. The SAGE system has the ability to analyze entire mRNA populations and to identify new genes (see box for details).
In this section, we present a SAGE application using
HotStarTaq DNA Polymerase to amplify the SAGE ditags.
SAGE (serial analysis of gene expression) starts with reverse transcription of an mRNA population. Uniformly sized, unique
“tags” are then generated from each cDNA using proprietary techniques. These tags are ligated in pairs to form “ditags”.
The ditags are then amplified by PCR and ligated into series of approximately 50 tags for sequencing. The overall transcription pattern is then analyzed using the SAGE software package.
The procedure amplifies each uniformly sized ditag with the same primers, avoiding artifacts due to variable PCR amplification encountered in differential display. Also, the SAGE process provides a permanent database of mRNA expression, without separate wet-lab procedures to identify each differentially displayed gene.
The SAGE process produces a single tag for each cDNA, allowing accurate quantitation of gene expression. The small
SAGE tags are long enough to uniquely identify a gene yet small enough to allow efficient, high-throughput sequencing.
By serial sequencing of the tags, the process enables analysis of approximately 50-fold more transcripts than comparable sequencing of single expressed-sequence tags (ESTs).
For more information about SAGE technology and a list of references, see the Genzyme website at: www.genzyme.com/sage/
M 1 2 3 4 5 6
Generation of a large quantity of pure, clean ditag is crucial for optimally linking together in subsequent steps of the SAGE procedure. As part of a study of gene-expression patterns in oral cancer, HotStarTaq DNA Polymerase was used to amplify SAGE ditags ( Figure 48 ). PCR with
HotStarTaq DNA Polymerase produced a clean, strong, reproducible band of 102 bp, which was not successful with the DNA polymerase from Supplier P
II previously used in this laboratory (data not shown).
Figure 48
SAGE ditags were amplified in the SAGE procedure in six experiments for 24 PCR cycles using HotStarTaq DNA Polymerase. The arrow indicates the 102 bp ditag band. M : 10 bp ladder.
(Data kindly provided by J. Llewelyn, Department of Craniofacial Development, Guy’s Hospital, King’s College London, UK.)
102 bp
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Prerequisites for successful PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Some general guidelines are given in Table1.*
Table1. General guidelines for standard PCR primers
Length:
GC content:
T m
:
Sequence:
18–30 nucleotides
40–60%
Simplified formula for estimating melting temperature ( T m
):
T m
= 2°C x (A+T) + 4°C x (G+C)
Whenever possible, design primer pairs with similar T m values.
Optimal annealing temperatures may be above or below the estimated T m
. As a starting point, use an annealing temperature 5°C below T m
.
• Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primer–dimer formation.
• Avoid runs of 3 or more Gs or Cs at the 3' end.
• Avoid a 3'-end T. Primers with a T at the 3' end have a greater tolerance of mismatch.
• Avoid complementary sequences within a primer sequence and between the primer pair.
• Commercially available computer software can be used for primer design.
Concentration: • Spectrophotometric conversion for primers:
Absorbance of 1 at 260 nm (1 A
260 unit) 20–30 µg/ml
• Molar conversions:
Primer length pmol/µg
18mer
20mer
168
152
20 pmol
119 ng
132 ng
Storage:
25mer
30mer
121
101
165 ng
198 ng
• Use 0.1–0.5 µM of each primer in PCR. For most applications, a 0.2 µM primer concentration will be sufficient.
Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/µl to avoid repeated thawing and freezing. Store all primer solutions at –20°C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.
* For further information see our guide Critical Factors for Successful PCR . To obtain a copy, visit the QIAGEN web site at www.qiagen.com or call one of the QIAGEN Technical Service Departments or local distributors listed on the last page.
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Occasionally, the exact nucleotide sequence of the target-template DNA will not be known, for instance when it has been deduced from an amino acid sequence. To enable such templates to be amplified by PCR, degenerate primers can be used.
These are actually mixtures of several primers whose sequences differ at the positions that correspond to the uncertainties in the template sequence.
Hot-start PCR often improves the specificity of PCR amplifications that employ degenerate primers by reducing the formation of nonspecific PCR products and primer–dimers. We recommend using HotStarTaq DNA Polymerase for highly specific amplification using degenerate primers (see page 7). Table 2 gives recommendations for further optimizing PCR using degenerate primers. Table 3 shows the codon redundancy of each amino acid.
Table 2. Guidelines for design and use of degenerate primers
Sequence: • Avoid degeneracy in the 3 nucleotides at the 3' end.
• If possible, use Met- or Trp-encoding triplets at the 3' end.
• To increase primer–template binding efficiency, reduce degeneracy by allowing mismatches between the primer and template, especially towards the 5' end (but not at the 3' end).
• Try to design primers with less than 4-fold degeneracy at any given position.
Concentration: • Begin PCR with a primer concentration of 0.2 µM.
• In case of poor PCR efficiency, increase primer concentrations in increments of 0.25 µM until satisfactory results are obtained.
Table 3. Codon redundancy
Amino acid
Met, Trp
Cys, Asp, Glu, Phe, His, Lys, Asn, Gln, Tyr
Ile
Ala, Gly, Pro, Thr, Val
Leu, Arg, Ser
Number of codons
3
4
1
2
6
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PCR products of up to 4 kb can be routinely amplified using standard PCR protocols. However, amplification of PCR products longer than 4 kb often fails without lengthy optimization. Reasons for failure are nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions, which have a greater effect on the amplification of longer
PCR products than on shorter ones.
The following guidelines may be helpful in amplifying products between 4 and 7 kb (referred to here as “long” PCR products) from complex templates using QIAGEN Taq DNA Polymerase, with its optimized PCR buffer and Q-Solution.
Table 4. General guidelines for amplifying long PCR fragments
PCR setup
• Run parallel PCR amplifications with and without Q-Solution.
• Perform a simplified hot start described in the QIAGEN Taq PCR Handbook .
• Try the following reaction mixture initially (optimization of primer concentration and template amount may be necessary):
1x QIAGEN PCR Buffer*
± 1x Q-Solution
0.2–0.5 µM each primer
200 µM each dNTP
2.5 U QIAGEN Taq DNA Polymerase
≥ 10 5 copies template DNA add water to give a final reaction volume of 50 µl.
Cycling conditions
Initial denaturation:
First 10 cycles
60 s 94°C
Denaturation:
Annealing:
10 s 92–94°C
60 s 50–68°C If the melting temperature of the primers is sufficiently high, a combined annealing/extension step may be performed at 68°C.
Extension: 60 s x EPL 68°C EPL is the expected product length in kb; e.g., for a 3.5 kb product, the extension step would be 3.5 x 60 sec = 210 sec.
Next 25 cycles
Denaturation:
Annealing:
Extension:
10 s 92–94°C
60 s 50–68°C If the melting temperature of the primers is sufficiently high, a combined annealing/extension step may be performed at 68°C.
(60 s x EPL)
+ 10 s each cycle
68°C
* Provides final concentration of 1.5 mM MgCl
2
PCR: longer PCR fragments
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HotStarTaq DNA Polymerase has been shown to successfully amplify a single-copy gene from just a single cell. The recommendations provided in Table 5 are intended to serve as a starting point for performing such a single-cell PCR from genomic template DNA. If the PCR product is undetectable or the product yield is too low, perform a nested PCR (see page 48).
Table 5. Recommendations for single-cell PCR
Isolation and • Single cells may be isolated by different methods (e.g., by flow cytometry or by micromanipulation). storage of • Keep samples cool during the cell-isolation procedure to prevent DNA degradation. single cells: • Transfer cell into a PCR tube that has been filled with 20 µl of
1x PCR Buffer. Immediately freeze the sample on dry ice.
• Store cell at –80°C until required for PCR analysis.
PCR setup: • Prepare a fresh master mix for single-cell PCR (see below).
• Thaw the cell on ice.
• Distribute 30 µl of the master mix into each PCR tube, and place the tubes in the thermal cycler. Immediately start the cycling program with a 10-min incubation step at 95°C to activate HotStarTaq DNA Polymerase.
50 cycles of PCR may be required to amplify a single-copy gene in one round of PCR.
Master mix Prepare a master mix that has a final volume of 30 µl per PCR, preparation: as detailed below.
Notes: • Addition of carrier nucleic acid is usually required (e.g., E.coli
5S rRNA).
• Use PAA gel- or HPLC-purified primers only.
Component
10x PCR Buffer*
25 mM MgCl
2
10 mM dNTP
Primer A
Primer B
5S ribosomal RNA ( E.coli
)
HotStarTaq DNA Polymerase
Distilled water
Single cell in 1x PCR Buffer
Total volume
Vol./reaction
3 µl
Variable
1 µl
Variable
Variable
Variable
1 µl
Variable
20 µl
50 µl
Final conc.
1x
–
200 µM of each dNTP
0.2 µM
0.2 µM
50 ng/reaction
5 units/reaction
–
–
–
* Contains 15 mM MgCl
2
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Multiplex PCR is a demanding PCR technique used for genetic screening, microsatellite analysis, and other applications where it is necessary to amplify several products in a single reaction. This technique often requires extensive optimization because primer–dimers and other nonspecific products may interfere with the amplification of specific products. Although primer–dimer formation can usually be avoided using hot-start PCR, amplification specificity is also influenced by other factors such as the PCR buffer and primer concentration. General guidelines are presented in Table 6 below.
Table 6. General guidelines for optimizing multiplex PCR
Step 1 Optimize cycling conditions for individual primer pairs
Prepare 50 µl reactions containing only one primer pair:
• 100–200 ng genomic DNA
• 0.2 µM each primer
• 1x QIAGEN PCR Buffer
• 1.25–2.5 units HotStarTaq DNA Polymerase
Adjust temperature and duration of annealing and extension steps until similar product yields are obtained with all primer pairs.
Step 2 Perform multiplex PCR
Use equimolar concentrations of all primer pairs and the optimized cycling conditions from step 1.
Step 3 Optimize multiplex conditions
Adjust the following parameters according to the guidelines below until the desired yield is obtained:
• Primer concentration of individual primer pairs in steps of 0.1–0.2 µM
• Annealing temperature in steps of 1°C
• Extension time in steps of 30 s
No product detected:
• Increase primer concentration
• Decrease annealing temperature
Low product yield:
• Increase primer concentration
• For short products, decrease extension time
• For long products, increase extension time
Excessive product yield:
• Decrease primer concentration
• For long products, decrease extension temperature to 68°C.
Step 4 If necessary, perform further optimization (to be used if steps 1–3 do not produce desired results)
• Use 1x Q-Solution*
• Use 1x Q-Solution* and decrease annealing temperature in steps of 1°C
*Q-Solution is a PCR additive provided with HotStarTaq DNA Polymerase.
PCR: multiplex PCR
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If PCR sensitivity is too low, a nested PCR method can increase PCR product yield. Nested PCR involves two rounds of amplification reactions. The first-round PCR is performed according to the PCR Protocol using HotStarTaq DNA Polymerase.
Subsequently, an aliquot of the first-round PCR product, for example 1 µl of a 1-in-10 3 –10 4 dilution, is subjected to a second round of PCR. The second-round PCR is performed with two new primers that hybridize to sequences internal to the first-round primer–target sequences. In this way, only specific first-round PCR products (and not nonspecific products) will be amplified in the second round. Alternatively, it is possible to use one internal and one first-round primer in the second PCR; this method is referred to as semi-nested PCR.
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Prerequisites for successful one-step RT-PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Some general guidelines are given in Table 7. The QIAGEN
OneStep RT-PCR Kit is designed to be used with gene-specific primers only. The use of random oligomers or oligo-dT primers is not recommended since this will result in the amplification of nonspecific products.
Table 7. General guidelines for standard RT-PCR primers
Length:
G/C content:
T m
:
Location:
Sequence:
18–30 nucleotides
40–60%
Simplified formula for estimating melting temperature ( T m
):
T m
= 2°C x (A+T) + 4°C x (G+C)
Whenever possible, design primer pairs with similar T m values.
Optimal PCR annealing temperatures may be above or below the estimated T m
. As a starting point, use an annealing temperature 5°C below T m
. Primer T m values should not be lower than the reversetranscription reaction temperature (e.g., 50°C).
• Design primers so that one half of the primer hybridizes to the 3' end of one exon and the other half to the 5' end of the adjacent exon (see Figure 49A, page 50). Primers will anneal to cDNA synthesized from spliced mRNAs, but not to genomic DNA. Thus, amplification of contaminating DNA is eliminated.
• Alternatively, RT-PCR primers should be designed to flank a region that contains at least one intron (see Figure 49B, page 50). Products amplified from cDNA (no introns) will be smaller than those amplified from genomic DNA (containing introns). Size difference in products is used to detect the presence of contaminating DNA.
• If only the mRNA sequence is known, choose primer annealing sites that are at least 300–400 bp apart. It is likely that fragments of this size from eukaryotic DNA contain splice junctions.
As explained in the previous point and Figure 49, such primers may be used to detect DNA contamination.
• Avoid complementarity of two or more bases at the 3' ends of primer pairs to reduce primer–dimer formation.
• Avoid mismatches between the 3' end of the primer and the target-template sequence.
• Avoid runs of 3 or more G or C nucleotides at the 3' end.
• Avoid a 3'- end T. Primers with a T at the 3' end have a greater tolerance of mismatch.
• Avoid complementary sequences within a primer sequence and between the primers of a primer pair.
• Commercially available computer software can be used for primer design.
RT-PCR: one-step RT-PCR primers
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Concentration:
Storage:
• Spectrophotometric conversion for primers:
1 A
260 unit ≡ 20–30 µg/ml
• Molar conversions:
Primer length
18mer
20mer pmol/µg
168
152
30 pmol (0.6 µM in 50 µl)
178 ng
198 ng
25mer
30mer
121
101
248 ng
297 ng
• Use 0.5–1.0 µM of each primer in one-step RT-PCR. For most applications with the QIAGEN
OneStep RT-PCR Kit, a primer concentration of 0.6 µM will be optimal.
Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing
10 pmol/µl to avoid repeated thawing and freezing. Store all primer solutions at –20°C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.
A Primer spans an intron/exon boundary
Genomic DNA mRNA
Exon Intron
RT-PCR
No product
Exon Exon Exon
RT-PCR
Product
B Primers flank an intron
Genomic DNA
Exon Intron
RT-PCR
Exon
Large product mRNA
Exon Exon
RT-PCR
Small product
Figure 49
50 RT-PCR: one-step RT-PCR primers
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Multiplex RT-PCR is a demanding amplification technique which allows the simultaneous detection of several RNA targets in a single reaction. Multiplex RT-PCR is used in applications such as analysis of chromosome translocations, detection of RNA virus serotypes, study of cytokine gene expression, and quantitative/semiquantitative RT-PCR.
Primers should be designed according to the guidelines given in Table 7, page 49. Table 8 provides further guidelines for optimization of multiplex RT-PCR conditions.
Table 8. Guidelines for multiplex RT-PCR
Step 1 Optimize cycling conditions for individual primer pairs
• Set up one-step RT-PCR samples according to the Protocol Using QIAGEN OneStep RT-PCR Kit using a primer concentration of 0.6 µM. Each reaction should contain only one primer pair.
• Determine one set of RT-PCR conditions (template amount, number of cycles, annealing temperature, and extension time) that produces satisfactory yield from each individual primer pair.
Step 2 Perform multiplex RT-PCR
Use 0.6 µM of each primer and the optimized cycling conditions from step 1.
Step 3 Optimize multiplex RT-PCR
• If the RT-PCR from step 2 results in different product yields, reduce the concentration of the primers yielding the most prominent RT-PCR product(s) in steps of 0.1 µM until all products are produced in similar quantities. Concentrations as low as 0.05 µM may be sufficient to amplify abundant transcripts.
• If altering the primer concentrations fails to improve the yield of long RT-PCR products, increase the extension time in increments of 30 s.
RT-PCR: multiplex RT-PCR
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The relative abundance of a transcript in different samples can be estimated by semiquantitative or relative RT-PCR. Typically, the signal from the RT-PCR product is normalized to the signal from an internal control included in all samples and amplified at the same time as the target. The normalized data from different samples can then be compared. Transcripts of housekeeping genes such as GAPDH or β -actin are frequently chosen as internal controls because they are abundantly expressed at relatively constant rates in most cells. However, the internal control transcript is usually more abundant than the transcript under study. This difference in abundance can lead to preferential amplification of the internal control and, in some cases, prevent amplification of the target RT-PCR product. Often, such problems can be overcome by reducing the internal-control primer concentration. The guidelines in Table 9 may be helpful in developing co-amplification conditions.
Table 9. Guidelines for co-amplification of an internal control
• Choose similar amplicon sizes for the target and the internal control but be sure that the products can be easily distinguished on an agarose gel.
• Determine RT-PCR conditions that are suitable for both amplicons by varying template amount, number of cycles, annealing temperature, and extension time.
• Initially, try primer concentrations of 0.6 µM for the target transcript and 0.3 µM for the internal control transcript.
• If the yield of internal standard greatly exceeds that of the specific target using the concentrations given above, reduce the internal-control primer concentration in steps of 0.05–0.1 µM. The optimal primer concentration for the internal control depends on the relative abundance and efficiency of amplification of the control and target transcripts. Control transcripts may be much more highly expressed than the target transcript. If the difference in abundance is too great, then RT-PCR of the internal control may interfere with the amplification of the target transcript.
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The QIAGEN OneStep RT-PCR Protocol is optimized for amplification of products of up to 2 kb. When amplifying RT-PCR products larger than 2 kb, it is recommended to use the modified cycling conditions described in Table 10.
Table 10. Modified thermal-cycler conditions for long RT-PCR products
Reverse transcription:
Initial PCR activation step:
30 min
15 min
Additional comments
45°C For RT-PCR products longer than 2 kb, a reverse-transcription reaction temperature of 45°C is recommended.
95°C HotStarTaq DNA Polymerase is activated by this heating step.
Omniscript and Sensiscript Reverse Transcriptases are inactivated and template cDNA is denatured.
3-Step cycling
Denaturation:
Annealing:
Extension:
Number of cycles:
Final extension:
10 s
0.5–1 min 50–68°C Approximately 5°C below T m of primers.
1 min x EPL
25–40
10 min
94°C
68°C EPL is the expected product length in kb; e.g., for a 3 kb product, the extension step would last 1 min x 3 = 3 min
68°C
The cycle number is dependent on the amount of template RNA and the abundance of the respective target.
RT-PCR: longer RT-PCR products
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Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/ chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step enzyme-catalyzed processes. QIAGEN offers a complete range of nucleic acid preparation systems, ensuring the highest-quality templates for PCR. Our innovative purification technologies allow streamlined and reproducible purification of nucleic acids. QIAGEN kits for DNA and RNA purification are based on three highly developed purification technologies: solid-phase anion-exchange, silica-gel–membrane, and hybrid-capture technology. QIAGEN silica-gel–membrane technology yields high-purity nucleic acids suitable for most molecular biology and clinical research applications, such as PCR and RT-PCR. For more sensitive applications, QIAGEN
Anion-Exchange Resin yields DNA or RNA of a purity and biological activity equivalent to at least two rounds of purification in
CsCl gradients, in a fraction of the time. For efficient hybrid capture of poly A + mRNA, QIAGEN Oligotex ® Resin consists of uniformly sized polystyrene–latex particles with covalently attached dC
10
T
30 oligonucleotides for superior poly A + mRNA-binding.
See Tables 11 and 12 below to select the system that is best for your needs. High-throughput and automated formats are also available for many kits. For more information about QIAGEN nucleic acid purification products, see the latest QIAGEN
Product Guide , visit our web site at www.qiagen.com
, or call QIAGEN Technical Services or your local distributor.
Table 11. DNA isolation
Genomic and viral DNA isolation
From animal tissues and cells
From clinical blood and tissue samples
From plants and fungi
High-molecular-weight genomic DNA isolation
Parallel genomic DNA and RNA isolation
DNeasy Tissue Kits
QIAamp DNA Kits
DNeasy Plant Kits
QIAGEN Genomic-tips
QIAGEN RNA/DNA Kits
Table 12. RNA isolation
Total RNA isolation
From animal cells and tissues, bacteria, yeast, and enzymatic reactions
From plants and fungi
From human whole blood
Viral RNA isolation
From cell-free body fluids
From animal cells and tissues (total RNA)
From plants and fungi (total RNA)
RNeasy Kits
RNeasy Plant Mini Kit
QIAamp RNA Blood Mini Kit
QIAamp Virus and QIAamp Viral RNA Kits
RNeasy Kits
RNeasy Plant Mini Kit
Poly A+ mRNA isolation
From total RNA or enzymatic reactions
Directly from animal cells and tissues
Parallel RNA and genomic DNA isolation
Small RNA (e.g., tRNA, 5S rRNA) isolation
Oligotex mRNA Kits*
Oligotex Direct mRNA Kits*
QIAGEN RNA/DNA Kits
QIAGEN RNA/DNA Kits
* Not available in Japan
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After amplification, PCR samples contain a complex mixture of specific PCR product and residual reaction components, such as primers, unincorporated nucleotides, enzyme(s), salts, mineral oil, and probably nonspecific amplification products. Before the specific PCR product can be used in subsequent experiments, it is often necessary to remove these contaminants. The
QIAquick system offers a quick and easy method for purifying the final PCR product. QIAquick PCR Purification Kits can also be used for fast cleanup of reverse transcription and other enzymatic reactions. For extraction of PCR products from gels, the
QIAquick or QIAEX ® II systems can be used. See Table 13 to choose the system that is best for your needs. For more information about QIAquick and QIAEX II products, see the latest QIAGEN Product Guide, visit our web site at www.qiagen.com
, or call QIAGEN Technical Services or your local distributor.
Table 13. DNA cleanup
PCR cleanup
Gel extraction
Of DNA fragments (70 bp –10 kb) from agarose gels
Of DNA fragments (40 bp – 50 kb) from agarose and polyacrylamide gels
QIAquick PCR Purification Kits
QIAquick Gel Extraction Kit
QIAEX II Gel Extraction Kit
Before and after PCR
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PCR
Allam, A., Kabelitz, D., and Hinz, T. (1998) Optimized RT-PCR of chimeric T-cell–receptor genes using HotStarTaq DNA
Polymerase. QIAGEN News 1998 No. 5, 1.
Löffert, D., Karger, S., Gundula, T., Ulber, V., and Kang, J. (1999) Optimization of multiplex PCR. QIAGEN News 1999 No. 2, 5.
Missel, A., Welters, S., Schmitz, A., and Löffert, D. (1999) Optimizing RT-PCR for greater specificity and reproducibility.
QIAGEN News 1999 No. 3, 1.
Ossendorf, M., and Prellwitz, W. (1998) Rapid and easy apolipoprotein E genotyping by restriction fragment analysis using two restriction enzymes and mutagenic primer for amplification. J. Lab. Med. 22 , 104.
Pring-Åkerbloom, P., Trijsenaar, F.E.J., Adrian, T., and Hoyer, H. (1999) Multiplex polymerase chain reaction for subgenus-specific detection of human adenoviruses in clinical samples. J. Med. Virol. 58 , 87.
Schwers, S., Ehlich, A., Kobsch, S., Missel, A., and Löffert, D. (2000) Single-cell PCR and RT-PCR: amplification specificity.
QIAGEN News 2000 No. 3, 14.
Aubourg, S. et al. (1999) The DEAD box RNA helicase family in Arabidopsis thaliana . Nucleic Acids Res. 27 , 628.
RT-PCR
Bosch, I., Melichar, H., and Pardee, A.B. (2000) Identification of differentially expressed genes from limited amounts of RNA. Nucl. Acids
Res. 28 , e27.
Burri, N., and Chaubert, P. (1999) Complex methylation patterns analyzed by single-strand conformation polymorphism.
BioTechniques 26 , 232.
Ciolino, H.P. and Yeh, G.C. (1999) The steroid hormone dehydroepiandrosterone inhibits CYP1A1 expression in vitro by a posttranscriptional mechanism. J. Biol. Chem. 274 , 35186.
Cornelison, D.D.W., and Wold, B.J. (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse muscle satellite cells. Devel. Biol. 191 , 270.
Horwitz, M., Benson, K.F., Person, R.E., Aprikyan, A.G., and Dale, D.C. (1999) Mutations in ELA2, encoding neutrophil elastase, define a
21-day biological clock in cyclic haematopoiesis. Nat. Genet. 23 , 433.
Imaizumi, T., et al. (2000) Expression of tumor necrosis factorα in cultured human endothelial cells stimulated with lipopolysaccharide or interleukin-1 α . Arterioscler. Thromb. Vasc. Biol. 20 , 410.
Korfhage, C., Fisch, E., Schröder-Stumberger, I., and Oelmüller, U. (2000) Solving difficult template problems in RT-PCR. QIAGEN News
2000 No. 1, 12.
Korfhage, C., Schröder-Stumberger, I., and Oelmüller, U. (2000) Maximizing primer specificity in reverse transcription. QIAGEN News
2000 No. 2, 12.
Léna, C., de Kerchove d’Exaerde, A., Cordero-Erausquin, M., Le Novère, N., del Mar Arroyo-Jimenez, M., and Changeux, J.-P. (1999) Diversity and distribution of nicotinic acetylcholine receptors in the locus ceruleus neurons. Proc. Natl. Acad. Sci. USA 96 , 12,126.
Martincic, D., Kravtsov, V., and Gailani, D. (1999) Factor XI messenger RNA in human platelets. Blood 94 , 3397.
Sharkey, M.E. et al. (2000) Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nat.
Med. 6 , 76.
Spiegel, M. et al. (1998) Pseudotype formation of Moloney Murine Leukemia virus with Sendai virus glycoprotein F. J. Virol. 72 , 5296.
56 References
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Taq DNA Polymerase — for standard and specialized PCR applications
Taq DNA Polymerase (250) 250 units Taq
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
DNA Polymerase,
Taq
Taq
DNA Polymerase (1000)
DNA Polymerase (5000)
4 x 250 units Taq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
20 x 250 units Taq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
Taq PCR Core Kit — for complete PCR setup
Taq PCR Core Kit (250) 250 units Taq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
, dNTP Mix †
Taq PCR Core Kit (1000) 4 x 250 units Taq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
, dNTP Mix †
Taq PCR Master Mix Kit — for convenient PCR setup
Taq
Taq
PCR Master Mix Kit (250)
PCR Master Mix Kit (1000)
3 x 1.7 ml Taq PCR Master Mix, ‡
3 x 1.7 ml distilled water
12 x 1.7 ml Taq PCR Master Mix, ‡
12 x 1.7 ml distilled water
HotStarTaq DNA Polymerase — for high-specificity hot-start PCR
HotStarTaq DNA Polymerase (250) 250 units HotStarTaq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
HotStarTaq DNA Polymerase (1000) 4 x 250 units HotStarTaq DNA Polymerase,
10x PCR Buffer,* 5x Q-Solution,
25 mM MgCl
2
HotStarTaq Master Mix Kit — the only ready-to-use solution for hot-start PCR
HotStarTaq Master Mix Kit (250)
HotStarTaq Master Mix Kit (1000)
3 x 0.85 ml HotStarTaq Master Mix † containing 250 units HotStarTaq DNA
Polymerase total, 2 x 1.7 ml distilled water
12 x 0.85 ml HotStarTaq Master Mix † containing 1000 units HotStarTaq DNA
Polymerase total, 8 x 1.7 ml distilled water
201443
201445
203203
203205
201203
201205
201207
201223
201225
203443
203445
* Contains 15 mM MgCl
2
† Contains 10 mM of each dNTP
‡ Provides a final concentration of 1.5 mM MgCl
2 and 200 µM of each dNTP
Ordering Information
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QIAGEN OneStep RT-PCR Kit — for highly efficient and simple one-step RT-PCR
QIAGEN OneStep RT-PCR Kit (25) For 25 reactions: QIAGEN OneStep
RT-PCR Enzyme Mix, 5x QIAGEN
OneStep RT-PCR Buffer,* dNTP Mix, †
5x Q-Solution, RNase-free water
QIAGEN OneStep RT-PCR Kit (100) For 100 reactions: QIAGEN OneStep
RT-PCR Enzyme Mix, 5x QIAGEN OneStep
RT-PCR Buffer,* dNTP Mix, †
5x Q-Solution, RNase-free water
Omniscript RT Kit — for reverse transcription using ≥ 50 ng RNA
Omniscript RT Kit (10) For 10 reverse-transcription reactions:
40 units Omniscript Reverse Transcriptase,
10x Buffer RT, dNTP Mix, ‡ RNase-free water
Omniscript RT Kit (50)
Omniscript RT Kit (200)
For 50 reverse-transcription reactions:
200 units Omniscript Reverse Transcriptase,
10x Buffer RT, dNTP Mix, ‡ RNase-free water
For 200 reverse-transcription reactions:
800 units Omniscript Reverse Transcriptase,
10x Buffer RT, dNTP Mix, ‡ RNase-free water
Sensiscript RT Kit — for reverse transcription using <50 ng RNA
Sensiscript RT Kit (50)
Sensiscript RT Kit (200)
For 50 reverse-transcription reactions:
Sensiscript Reverse Transcriptase,
10x Buffer RT, dNTP Mix, ‡ RNase-free water
For 200 reverse-transcription reactions:
Sensiscript Reverse Transcriptase,
10x Buffer RT, dNTP Mix, ‡ RNase-free water
210210
210212
205110
205111
205113
205211
205213
* Contains 12.5 mM MgCl
2
† Contains 10 mM of each dNTP
‡ Contains 5 mM of each dNTP
58 Ordering Information
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Argentina
Tecnolab S.A.
Charlone 144 - 1427
Capital Federal
Tel: (011) 4555 0010
Fax: (011) 4553 3331
E-mail: info@tecnolab.com.ar
Web site: www.tecnolab.com.ar
Czech Republic
BIO-CONSULT spol. s.r.o.
Boz˘ejovická 145
142 01 Praha-Libus˘
Tel: (02)4447 1239
Fax:
E-mail:
(02)47 29 792 bio-cons@login.cz
Web site: www.bio-consult.cz
Austria/Hungary/Slovenia
R. u. P. MARGARITELLA
Ges. m.b.H.
BIOTRADE
Breitenfurter Straße 480
1230 Wien-Rodaun
Austria
Tel:
Fax:
E-mail:
(01) 889 18 19
(01) 889 18 19 20
BIO-TRADE@TELECOM.AT
Denmark
Merck Eurolab A/S
Roskildevej 16
2620 Albertslund
Tel: 43 86 87 88
Fax:
E-mail:
43 86 88 89 info@merckeurolab.dk
Web site: www.merckeurolab.dk
Belgium/Luxemburg
Westburg b.v.
P.O. Box 214
3830 AE Leusden
The Netherlands
Tel:
Fax:
0800-1-9815
(31) 33-4951222
E-mail: info@westburg.nl
Web site: www.westburg.nl
Egypt
Clinilab
P.O. Box 12 El-Manial
4, 160 St., El-Etehad Square
Riham Tower, El-Maadi
Cairo
Tel:
Fax:
E-mail:
525 7212
525 7210
Clinilab@intouch.com
Brazil
Labtrade do Brazil
Av. Barão do Rego Barros, 542
V. Congonhas
Cep: 04612-041
São Paulo-Brasil
Tel:
Fax:
(11) 543 1455 or 0800 551321
(11) 531 3210
E-mail: labtrade@sol.com.br
Web site: www.labtrade.com.br
Central & South America
Labtrade Inc.
6157 NW 167th Street F-26
Miami, FL 33015
USA
Tel:
Fax:
(305) 828-3818
(305) 828-3819
E-mail: labtrade@icanect.net
Web site: www.labtrade.com
Finland
Merck Eurolab Oy
Niittyrinne 7
02270 Espoo
Tel:
Fax:
E-mail:
Web site: www.merckeurolab.fi
Greece
BioAnalytica S.A.
11, Laskareos Str.
11471 Athens
Tel:
Fax:
E-mail:
(01)-640 03 18
(01)-646 27 48 bioanalyt@hol.gr
India
(09)-804 551
(09)-804 55200 info@merckeurolab.fi
Genetix
C-88, Kirti Nagar
Lower Ground Floor
New Delhi-110 015
Tel: (011)-542 1714
Fax:
E-mail: or (011)-515 9346
(011)-546 7637 genetix@vsnl.com
China
Gene Company Limited
Unit A, 8/F., Shell Industrial Building
12 Lee Chung Street
Chai Wan, Hong Kong, P.R.C.
Tel:
Fax:
E-mail:
(852)2896-6283
(852)2515-9371
Hong Kong: info@genehk.com
Beijing: gene@public2.bta.net.cn
Shanghai:
Chengdu:
Guangzhou: gene@public.sta.net.cn
gene@public.cd.sc.cn
gzyitao@public.guangzhou.gd.cn
Israel
Westburg (Israel) Ltd.
1, Habursekai St. Kiriat Ha'asakim
Beer Sheva 84899
Tel: 07. 66 50 814
Fax:
E-mail: or 1-800 20 22 20 (toll free)
07. 62 77 019 info@westburg.co.il
Web site: www.westburg.co.il
Cyprus
Scientronics Ltd
34, Zenonos Sozou Str.
1075 Lefkosia
Phone:
Fax:
E-mail:
02-765 416
02-764 614 sarpetsa@spidernet.com.cy
Korea
LRS Laboratories, Inc.
SongBuk P.O. Box 61
Seoul, 136-600
Tel: (02) 924-86 97
Fax:
E-mail:
(02) 924-86 96 lrslab@nownuri.net
Malaysia
Research Biolabs Sdn. Bhd.
79A Jalan SS15/4C
Subang Jaya
47500 Petaling Jaya, Selangor
Tel:
Fax:
E-mail:
(03)-7312099
(03)-7318526 biolabs@tm.net.my
South Africa
Southern Cross Biotechnology (Pty) Ltd
P.O. Box 23681
Claremont 7735
Cape Town
Tel:
Fax:
(021) 671 5166
(021) 671 7734
E-mail: info@scb.co.za
Web site: www.scb.co.za
Mexico
Quimica Valaner S.A. de C.V.
Jalapa 77, Col Roma
Mexico D.F. 06700
Tel:
Fax:
(5) 525 57 25
(5) 525 56 25
E-mail: qvalaner@infosel.net.mx
Spain
IZASA, S.A.
Aragón, 90
08015 Barcelona
Tel: (93) 902.20.30.90
Fax: (93) 902.22.33.66
The Netherlands
Westburg b.v.
P.O. Box 214
3830 AE Leusden
Tel: (033)-4950094
Fax: (033)-4951222
E-mail: info@westburg.nl
Web site: www.westburg.nl
Sweden
Merck Eurolab AB
Fagerstagatan 18A
16394 Sp°anga
Tel:
Fax:
E-mail:
(08) 621 34 00
(08) 760 45 20 info@merckeurolab.se
Web site: www.merckeurolab.se
New Zealand
Biolab Scientific Ltd.
244 Bush Road
Albany, Auckland
Tel: (09) 980 6700 or 0800 933 966
Fax: (09) 980 6788
E-mail: info@biolab.co.nz
Web site: www.biolab.co.nz
Taiwan
TAIGEN Bioscience Corporation
3F. No. 306, Section 4, Chen-Der Road
Taipei
Taiwan, R.O.C.
Tel: (02) 2880 2913
Fax:
E-mail:
(02) 2880 2916 taigen@ms10.hinet.net
Norway
Merck Eurolab AS
Postboks 45, Kalbakken
0901 Oslo
Kakkelovnskroken 1
Tel: 22 90 00 00
Fax:
E-mail:
22 90 00 40 info@merckeurolab.no
Web site: www.merckeurolab.no
Thailand
Theera Trading Co. Ltd.
64 Charan Sanit Wong Road
(Charan 13) Bangkokyai
Bangkok 10600
Tel:
Fax:
E-mail:
(02) 412-5672
(02) 412-3244 theetrad@samart.co.th
Poland
Syngen Biotech Sp.z.o.o.
ul.Legnicka 62 A
54-204 Wroclaw
Tel:
Fax:
(071) 351 41 06 or 0601 70 60 07
(071) 351 04 88
E-mail: info@syngen.com.pl
Web site: www.syngen.com.pl
QIAGEN Importers
Saudi Arabia
Abdulla Fouad Co. Ltd.
Medical Supplies Division
Prince Mohammed Street
P.O. Box 257, Dammam 31411
Kingdom of Saudia Arabia
Tel: (03) 8324400
Fax:
E-mail:
(03) 8346174 sadiq.omar@abdulla-fouad.com
Portugal
IZASA PORTUGAL, LDA
Rua do Proletariado, 1 - Quinta do Paizinho
2795-648 Carnaxide
Tel:
Fax:
(1)-424 73 64
(1)-417 26 59
All other countries
QIAGEN GmbH, Germany
Singapore
Research Biolabs Pte Ltd.
29 Lucky Crescent
Singapore 467742
Tel: 445 7927
Fax:
E-mail:
448 3966
BIOLABS@SINGNET.COM.SG
Slovak Republic
BIO-CONSULT Slovakia spol. s.r.o.
Ruz˘ová dolina 6
SK-821 08 Bratislava
Tel/Fax: (07) 50 221 336
E-mail: bio-cons@post.sk
Web site: www.bio-consult.cz
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