application note
728
nucleic acid amplification and analysis
Use of amplified DNA as a substrate
for multiplex PCR
GenomiPhi DNA Amplification Kit
Key words: GenomiPhi DNA Amplification Kit, genomic DNA,
multiplex, PCR, real-time PCR
Other materials
• PicoGreen™ dsDNA Quantitation Kit (Molecular Probes Inc)
GenomiPhi™ DNA Amplification Kit utilizes a novel technology to perform representative whole genome amplification
from small amounts of genomic DNA. The method utilizes
Phi29 DNA polymerase to exponentially amplify single- or
double-stranded linear DNA templates by strand displacement. This simple, isothermal method produces microgram
quantities of DNA from as little as 1 ng of starting material
in an overnight reaction. The majority of amplified DNA is
greater than 10 kb in length, and the proofreading activity
of Phi29 DNA polymerase preserves the original genetic
sequence.
The amplified DNA from GenomiPhi Kit can substitute
for genomic DNA in many downstream applications. Since
many genomic DNA applications begin with PCR amplification of a specific fragment, we have extensively characterized GenomiPhi Kit amplified DNA as a substrate for PCR.
Multiplex PCR from crude amplified DNA may be less
efficient, especially for larger amplicons in the multiplex.
However, this application note demonstrates that multiplex
performance is fully recovered by purifying the GenomiPhi
Kit product or by adding common PCR enhancers.
Products used
GenomiPhi DNA Amplification Kit
25-6600-01
DNA Polymerization Mix
27-2094-02
Stock dNTPs (100 mM each)
27-2035-01
MicroSpin™ G-50 columns
27-5330-01
100 Base-Pair Ladder
27-4007-01
• 10x PCR Buffer II, 10 mM Tris-HCl pH 8.3, 50 mM KCl
(PerkinElmer Biosystems)
• GeneChip™ reagent kit (Affymetrix Inc)
• GeneChip CYP450 primer set (Affymetrix Inc)
• AmpliTaq Gold™ DNA Polymerase (PerkinElmer Biosystems)
• human DNA, female (Promega)
• human DNA, male (Promega)
Protocol
1
●
DNA amplification
Note: Reactions must be prepared on ice to prevent non-specific
enzymatic activity by Phi29 polymerase.
1.1 Mix 1 µl (10 ng) human genomic DNA with 9 µl GenomiPhi
Sample Buffer.
1.2 Denature at 95 ºC for 3 min, and then cool to 4 ºC by
placing on ice.
Note: Do not denature for longer than 3 min as it can cause nicking
of the DNA.
1.3 Add 9 µl GenomiPhi Reaction Buffer and 1 µl GenomiPhi
Enzyme Mix.
Note: A master mix of GenomiPhi Reaction Buffer and Enzyme Mix
can be prepared just prior to addition. Do not prepare master mix
in advance.
× multiplex buffer
10×
1.4 Incubate at 30 ºC for 16–18 h.
(10 mM Tris PH 8.0, 0.1% Tween™ 20, and 37.5 µg/µl BSA)
1.5 Denature at 65 ºC for 10 min, and then place on ice.
1.6 Quantitate amplification yield by PicoGreen assay according to
the GenomiPhi DNA Amplification Kit protocol book.
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nucleic acid amplification and analysis
1125
1125
878
762
Fig 1. Simplex PCR reactions
were performed with each of
the seven primer pairs using
either unpurified GenomiPhi Kitamplified DNA or genomic DNA
as templates. Marker lanes
contain 100 bp ladders. Each
PCR primer pair amplifies
robustly from either template.
878
762
444
444
159
250
159
250
171
171
GenomiPhi
Genomic DNA
Optimization methods
bp
1125
Fig 2. Human DNA was amplified with GenomiPhi DNA
Amplification Kit and used as template along with genomic
DNA in a multiplex PCR. In lane 1, the 1125 and 878 bp products in the multiplex PCR were under-amplified. 10x multiplex
buffer was added to the reaction in lane 2.
878
762
444
250
178/159
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genomic DNA without buffer
100 Base-Pair Ladder
A 10× multiplex buffer is formulated from 10 mM Tris
pH 8.0, 0.1% Tween 20, and 37.5 µg/ml BSA. Addition
of this buffer to multiplex PCR reactions using unpurified
GenomiPhi Kit DNA products improves the performance
of the multiplex PCR (Fig 2).
unpurified amplified template with buffer
Addition of enhancers improves
multiplex performance
unpurified amplified template
This application note describes optimization methods
for multiplex PCR reactions using DNA amplified with
GenomiPhi Kit. For this application note, the multiplex
PCR is a seven-plex reaction using the GeneChip CYP450
primer set. The primers shown in Figure 1 are used for
the multiplex reactions described below.
1
2
3
4
nucleic acid amplification and analysis
Additional Taq polymerase or purification
of GenomiPhi material also improves
multiplex performance
The performance of GenomiPhi amplified DNA in multiplex
PCR was improved by adding more Taq DNA polymerase
to the PCR reaction or by purifying the amplified DNA
prior to PCR. Two simple purification methods—gel filtration on MicroSpin G-50 spin columns or ethanol precipitation
with sodium acetate/EDTA (data not shown)—worked equally
well (Fig 3).
Conclusions
DNA amplified with GenomiPhi Kit performs identically to
genomic DNA in multiplex PCR when one of the following
modifications is followed:
• Add Tween 20 and BSA to the multiplex PCR.
3
purified amplified DNA
2
added enzyme (10 U)
genomic DNA
1
added enzyme (5 U)
100 Base-Pair Ladder
• Purify the GenomiPhi Kit amplified DNA template using a spin
column or sodium acetate/EDTA and ethanol precipitation.
2x PCR primer
unpurified amplified DNA
• Add more enzyme to the multiplex PCR.
4
5
6
7
bp
1125
878
762
444
250
178/159
Fig 3. When GenomiPhi Kit amplified DNA template is purified
prior to PCR or if more Taq polymerase is added to the reaction,
performance is comparable to genomic DNA template.
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nucleic acid amplification and analysis
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Amersham Biosciences UK Limited
Amersham Place, Little Chalfont, Buckinghamshire, England HP7 9NA
GenomiPhi and MicroSpin are trademarks of Amersham Biosciences Ltd
Amersham and Amersham Biosciences are trademarks of Amersham plc
PicoGreen is a trademark of Molecular Probes Inc
Amersham Biosciences AB
SE-751 84 Uppsala, Sweden
AmpliTaq Gold is a trademark of Applied Biosystems Inc
GeneChip is a trademark of Affymetrix Inc
Tween is a trademark of ICI Americas Inc
Amersham Biosciences Corp.
800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA
Amersham Biosciences GmbH
Munzinger Strasse 9, D-79111 Freiburg, Germany
Amersham Biosciences (SV) Corp.
928 East Arques Avenue, Sunnyvale CA 94085 USA
The GenomiPhi DNA Amplification Kit and the use thereof for DNA synthesis is covered
by US patent application number 09/920,571 and US patents 5,854,033, 5,198,543,
5,576,204 and 5,001,050 license exclusively to Amersham Biosciences Corp.
The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular
Systems and F Hoffmann-La Roche Ltd. A license to use the PCR process for certain
research and development activities accompanies the purchase of certain reagents from
licensed suppliers such as Amersham Biosciences UK Limited and Affiliates when used
in conjunction with an authorized thermal cycler.
© Amersham Biosciences Corp. 2003 – All rights reserved
All goods and services are sold subject to the terms and conditions of sale of the company
within the Amersham Biosciences group which supplies them. A copy of these terms and
conditions is available on request.
www.amershambiosciences.com
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