Biochemistry 611
Nucleic Acids
8-28-07
Chad Wilkerson
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Post-doctoral fellow in Kevin Sarge’s lab
Dept. Biochemistry, BBSRB Building
Lab phone 257-7349
Email: dcwilk2 @ uky.edu
Topics To Be Covered
• Isolating
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Dissociation/deproteinization
Precipitation
• Quantitating
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UV absorption
• Separating
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Gel electrophoresis: Agarose & Polyacrylamide
• Analysis
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DNA: Southern blot, gel shift (EMSA), DNase footprinting,
ChIP, Promoter pull-down, PCR
RNA: RT-PCR, RACE, Exon trapping, PCR-based cDNA cloning,
RNase Protection, northern blot, nuclear run-off, primer extension
Isolation of Nucleic Acids
Two Main Steps for Isolation
1)
2)
Dissociation/deproteinization
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detergent (e.g. SDS, Triton X-100, NP40, CHAPS)
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An organic (e.g. phenol)
DNA  phenol:chloroform:isoamyl alcohol (25:24:1) at pH 8.0
RNA  acidic pH (below 7) DNA will denature and partition into the organic phase
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Strong electrolyte (e.g. guanidinium isothiocyanate –Trizol and RNA Stat-60)
Precipitation
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Raising the salt concentration to at least 0.1M and adding an alcohol (67% ethanol or 50%
isopropanol) precipitates nucleic acids from the aqueous phase
Common salts include: sodium acetate (NaAc) – samples brought to 0.3M
potassium acetate (KAc) – samples brought to 0.3M
ammonium acetate (NH4Ac) – samples brought to 2M
Isolation of Nucleic Acids
Things to keep in mind when isolating nucleic acids
1) The integrity of the nucleic acid
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low and high pH can lead to hydrolysis of nucleic acids
Excess pipetting or vortexing can shear DNA
2) Any enzyme requirements
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Specific salts and salt concentrations can inhibit enzymes
EDTA can inhibit reactions
3) Any functional requirements
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Some technologies require higher purification of nucleic acids
Isolation of Nucleic Acids
Additional topics related to isolation of nucleic acids
1) Tissue disruption
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Dounce homogenizer
Mortar and pestle
Sonication
2) Cellular fractionation
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Examples: nuclei, mitochondria, polysomes
3) Chromatographic purifications
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Examples: CsCl gradients, DEAE cellulose, oligo dT cellulose
Quantitating Nucleic Acids
Definition of O.D. at A260 refers to the O.D. reading when the sample in question is diluted to
1.0ml of ddH20 and read in a 1cm quartz cuvette at 260nm.
Nucleic Acids absorb UV light at a maximum of 260nm
There is a direct relationship between the concentration of a nucleic acid
and its absorption of UV light at 260nm
40 x OD260 of sample = concentration of RNA (ug/mL)
50 x OD260 of sample = concentration of DNA (ug/mL)
33 x OD260 of sample = concentration of oligonucleotide (ug/mL)
1 A260 dsDNA = 50ug
1 A260 ssDNA = 33ug
1 A260 ssRNA = 40ug
Quantitating Nucleic Acids
The relative purity of nucleic acid samples can be determined by measuring
their absorption at other wavelengths.
2 main contaminates include proteins and polysaccarides which have absorption
maximas at 280nm and 230nm respectively.
An uncontaminated RNA sample would have a 230, 260, 280 ratio of 1:2:1
An uncontaminated DNA sample would have a ratio of 1:1.8:1
Separating Nucleic Acids
Gel Electrophoresis
Agarose
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Less analytical
Typically used to separate nucleic acids greater than 100 bp
Concentrations range from 0.4% - 3%
Buffers commonly used include TAE or TBE (non-denaturing) and MOPS-formaldehyde
(denaturing)
Polyacrylamide
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High resolution capacity
Concentrations range from 4% - 20%
Buffers commonly used include TBE or TTE (Tris-taurine EDTA)
For denaturing nucleic acids urea is added to a final concentration of 7-8M
Separating Nucleic Acids
Agarose
Polyacrylamide
Agarose (%)
Effective Range of
Separation of Linear
DNA molecules (kb)
0.3
60-5
3.5
100 - 1000
0.6
20 – 1
5.0
80 - 500
0.7
10 – 0.8
8.0
60 - 400
0.9
7 – 0.5
12.0
40 - 200
1.2
6 – 0.4
20.0
10 - 100
1.5
4 – 0.2
2.0
3 – 0.1
Acrylamide (%)
Effective Range of
Separation (nucleotides)
Analysis of Nucleic Acids
DNA Analysis
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Southern blot
PCR
DNA:Protein Interactions
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Gel shift (EMSA)
DNase footprinting
Chromatin immunoprecipitation (ChIP)
Promoter pull- down
RNA Analysis
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RT-PCR
Race and Exon Trapping
PCR based cDNA cloning
northern blot
RNase Protection
Primer extension
Nuclear run-off
Southern Blot
Southern Blot (named after Edward M. Southern)
Commonly used to determine the molecular weight of a restriction fragment, to measure
relative amounts in different samples and to locate a particular sequence of DNA within a
complex mixture
Basic Protocol:
1) Fragment DNA using restriction enzymes
2)
Separate fragments by agarose gel electrophoresis
3)
DNA fragments are denatured and transferred to
a nitrocellulose membrane
4)
These membrane-bound fragments are assayed
for their ability to hybridize with a specific labeled
nucleotide sequence (probe).
Probes:
• Range in size from small (16 mers) to very
large (500+) DNA fragments
• Labeled at their terminus through kinase treatment
or internally through nick translation
• Labels can be in the form of isotopic or
chromogenic
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
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Invented in 1983 by Kary Mullis – Nobel Prize in Chemistry 1993
Allows the rapid amplification of DNA
Core components include:
Template
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Purity, source, concentration
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genomic DNA ~ 100-250 ng
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plasmid DNA ~ 20 ng
Critical Parameter:
Annealing Temperature
• About 5-7°C below Tm of primer pairs
Buffer
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MgCl2 necessary
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(0.5mM to 3.0mM  1.5mM default)
Annealing Time
• Rule of thumb is 1kb per minute
dNTP’s
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Final conc 200M - too high can inhibit rxn
Primer Design
• Discussed later
Polymerase
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Error rate and Conditions (next slide)
Primer(s)
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Size (typically 18-30 nt)
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G+C content (40-60%)
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Minimize secondary structure (hairpins)
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Concentration (0.1 and 0.5 mM)
LOTS OF POLYMERASES
Taq Polymerase
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Isolated from Thermus aquaticus in 1976
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Catalyze template-directed synthesis of DNA from nucleotide triphosphates
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Requires a primer having a free 3' hydroxyl is required to initiate synthesis
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Magnesium ion is necessary
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Has a maximal catalytic activity at 70 to 80 °C (optimal is 72°C)
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Incorporates approx. 125,000 nucleotides before making an error
Other themostable polymerases
Pfu: Pyrococcus furiosus
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Lowest error rate of known thermophilic polymersases
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Incorporates approx. 767,000 nucleotides before making an error
Vent (or Ttl): Thermococcus litoralis
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The most heat stable of all (halflife of 7 h at 95°C)
Tgo: Thermus aquaticus
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Highly processive = copies fast
Tth: Thermus thermophilus
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Copies long sequences
Primer Design:
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Size (typically 18-30 nt)
G+C content (40-60%)
Minimize secondary structure →
Concentration (0.1 and 0.5 mM)
Avoid runs of 3 or more G or C at the 3' end
Avoid a T at the 3' end
Avoid mismatches at the 3' end
Avoid complementary sequences within a primer and between primers
Melting Temperature (Tm):
Tm by definition is the temperature in which ½ the molecules in a hybridizing pair are single
stranded
Calculating the Tm:
1) 2 + 4 rule
2) Software: Primer Premiere
3) Online: IDTDNA.com
4) Trial and error
Primer Design:
Calculating the Tm using the 2+4 rule:
TACCTAGGTTGACCATCTACTAA
TACCTAGGTTGACCATCTACTAA
TACCTAGGTTGACCATCTACTAA
=
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9 G+C
14 A+T
Tm = 2°C x (14) + 4°C x (9)
Tm = 28°C + 36°C = 64°C
Hsp90 Set 4 Primers
50°
500bp
300bp
200bp
100bp
54°
58°
Hsp90 Set 5 Primers
60°
50°
62
54°
65
58° 68
60°
Types of PCR:
Real-time PCR
• More quantitative than conventional PCR
• Measurements are taken early in reaction rather than at the end point as in
conventional PCR
RT-PCR
• Makes cDNA from RNA
Nested-PCR
• Consists on two consecutive PCR reactions
• The amplified product from the first reaction acts as template DNA for the second
• ** See Supplement online
Hot-start PCR
• Reaction starts at 98°C without a slow warm up
• Primers do not have the chance to anneal at temperatures lower than the Tm
• Amplified products tend to be cleaner
Touchdown PCR
• PCR cycling begins at annealing temp above the expected annealing temp
• The annealing temp is decreased every 1-3 cycles until it reaches the expected annealing temp
Real-Time PCR
Based on detecting and quantifying the fluorescence of a reporter
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon
production at each PCR cycle (in real time) as opposed to the endpoint detection
Three general methods for the quantitative detection:
1. DNA-binding agents (SYBR Green)
2. Hydrolysis probes (TaqMan, Beacons, Scorpions) – utilizes exonuclease activity of polymerase!
3. Hybridization probes (Light Cycler)
More than you would ever want to know: http://www.dorak.info/genetics/realtime.html
The fluorescent signal increase in direct proportion to the amount of PCR product in a reaction.
By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR
reaction during exponential phase where the first significant increase in the amount of PCR
product correlates to the initial amount of target template.
Real-time PCR advantages
not influenced by non-specific amplification
amplification can be monitored real-time
no post-PCR processing of products (no gel analysis, low contamination risk, less loss)
rapid cycling (30 minutes to 2 hours)
range of detection is as low as a 2-fold change up to 1010-fold
requirement of 1000-fold less RNA than conventional assays
confirmation of specific amplification by melting point analysis
not much more expensive than conventional PCR (except equipment cost)
→
Different dilutions of the same template
Increasing
Fluorscence
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# PCR CYCLES
What are the more common techniques used
to study protein:DNA interactions?
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Gel shift (EMSA)
DNase footprinting
Chromatin immunoprecipitation (ChIP)
Promoter pull-down
Gel Shift or Electrophoretic Mobility Shift Assay
(EMSA)
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Assay provides a simple and rapid method for detecting in vitro interactions between
DNA and proteins
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Commonly used to study sequence-specific DNA-binding proteins such as
transcription factors
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The assay is based on the observation that complexes of protein and DNA migrate
through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or
double-stranded oligonucleotides
Binding Reaction:
Protein/Extract
Labeled Probe
Buffer
Antibody **
Competitor DNA **
Excess unlabeled
oligonucleotide
Antibody to DB Protein
DB Protein
Labeled oligonucleotide
DNase Footprinting
• The method of choice for identifying sequence specific binding of proteins to DNA
• Developed in 1978 by Galas and Schmitz
Footprint
Look for papers on Biochemistry website:
Galas_and_Schmitz_Footprinting.pdf
Kang_Footprinting.pdf
Chromatin Immunoprecipitation Assay (ChIP)
Chromatin immunoprecipitation (ChIP) is a powerful in vivo method to show
interaction of proteins associated with specific regions of the genome.
ChIP allows you to detect recruitment of a particular transcription factor to a
promoter region, analyze the interaction of any protein with any DNA sequence in vivo.
Fragments of DNA purified by ChIP can be used for cloning (i.e. Farnham paper)
More information can be found at: http://www.upstate.com/chip
and Farnham_ChIP_Cloning.pdf (Biochemistry website)
Purified DNA ready
to be assayed (i.e. PCR)
Protein bound DNA within nuclei
(only nuclei shown)
Crosslink DNA+Proteins
Isolate and lyse nuclei
Purify DNA
Shear DNA
– sonication most common method
Reverse crosslink by incubating
at 67°C with 200mM NaCl
Add antibody against protein of
interest and IP protein+DNA complex
Wash extensively with various salt buffers and
release antibody from protein+DNA complexes
with elution buffer (SDS+NaHCO3)
Promoter
Pull-down
Lyse Cells
Technique to identify proteins
that bind to a specific DNA
sequence
Mixture of
Proteins
Agarose bound Promoter
Region Fragment
Protein/DNA complex
 Complex can be purified by centrifugation
Release
Assay Purified
Proteins
Proteomic Identification
via Mass Spectroscopy
Proteomic Identification
via Western Blot
RNA Analysis
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RT-PCR
PCR based cDNA cloning
northern blot
RNase Protection
Primer extension
Nuclear run-off
Race and Exon Trapping
Reverse Transcriptase-PCR (RT-PCR)
Technique used to make cDNA from RNA
Template: RNA
Two consecutive reactions
• Reaction #1  Reverse transcription of RNA into cDNA (RNA:DNA hybrid)
• Reaction #2  Standard PCR reaction to make double stranded cDNA
Most Common Uses:
• Looking at gene expression (mRNA levels)
• Assaying viral systems
RT-PCR
Basic Reaction Mixture:
RNA
dNTPs
Primers
1x Buffer
Reverse Transcriptase
RNase Inhibitor
Thermophilic Polymerase
PCR based
cDNA Cloning
Commonly used to make a cDNA
library from mRNA
Northern Blots
Similar to southern blots in that it involves the separation of RNA species on
agarose gels and their transfer to nitrocellulose. Unlike Southern blots, Northern
blots are separated on a denaturing formaldehyde-agarose gel and gels are not
treated with NaOH prior to transferring to nitrocellulose.
Nuclease Protection Assay (NPA)
Nuclease protection assays (NPAs) include both ribonuclease protection assays
(RPAs) and S1 nuclease assays
These two assays are an extremely sensitive method for the detection, quantification
and mapping of specific RNAs in a complex mixture of total cellular RNA.
There are several advantages to this technique including (1) multiple mRNAs can be
assayed in a single RNA preparation (2) the length of each gene fragment is unique
allowing multiple probes to be synthesized together and hybridized to a single target
sample (3) highly specific and sensitive assay allowing the detection of sub-picograms
quantities of specific mRNA
The basis method involves:
1) Hybridize in solution a single-stranded antisense probe(s) to an RNA sample
2) After hybridization, any unhybridized probe and sample RNA are removed by
digestion with nucleases
3) The nucleases are inactivated and the remaining probe:target hybrids are precipitated.
4) These products are separated on a denaturing polyacrylamide gel and are visualized
Detailed Information can be found at:
http://www.ambion.com/techlib/basics/npa/
RNase Protection Assay
What in the world would you use this for??
Example:
You knockout a transcription factor in a mouse. You want to know if the lack of this
transcription factor affects the transcription of gene X, gene Y, and gene Z.
You can probe for the presence of the mRNA for each of the genes in question using
RNase Protection Assays
Primer Extension
Primer extension is used to map the 5' ends of DNA or RNA fragments.
Basic Protocol:
1. A specific oligonucleotide primer is labeled, usually at
its 5' end, with 32P
2. The labeled primer is annealed to a position
downstream of that 5' end of the template
3. The primer is extended with reverse transcriptase
(making a fragment that ends at the 5' end of the template).
DNA polymerase can also be used with DNA templates.
4. The newly synthesized labeled fragment is analyzed by gel
electrophoresis
What in the world would you use this for??
1. Can identify the transcription start site
2. RPA can tell you if a mRNA species is present
but primer extension can provide sequence size
For more information see:
http://www.promega.com/tbs/tb113/tb113.pdf
Nuclear Run-off Assays
• Sensitive method for measuring rates of expression (transcription) of a specific gene
• Based on incorporation of radiolabeled NTPs into elogating mRNAs and counting the
radioactivity
General Protocol:
1)
2)
3)
4)
5)
6)
Isolate nuclei
Incubate with 32P-UTP
Treat with DNase
Hybridize to denatured-immobilized cDNA corresponding to the mRNA
Treat with RNase
Count radioactivity
Biochemistry Website:
Baldassare_NRO.pdf
Li_Chaikof_NRO.pdf
Nuclear Runoff Assay
Endotoxin (stimulates Tc)
Imidazole
Assaying the effect of SB203580 (imidazole) on IL-1 (cytokine) gene transcription in RAW264.7 cells
Raw264.7 cells were stimulated with LPS (endotoxin from E. coli) in the presence or absence of
SB203580 at the indicated concentrations and analyzed by nuclear run-on analysis
Equal cpm of radiolabeled run-on RNA were used to probe individual nylon strips carrying an
excess of the indicated denatured cDNA probes. The Bluescript plasmid (BS) was included as a
background control because the murine IL-1 and IL-1ß, and TNF- cDNAs were all subcloned into
this plasmid. The blots were exposed for 2–3 wk, and the resultant films were scanned and digitized
on a PhosphorImager. Shown are representative data from four separate similar experiments.
Conclusion: the imidazole does inhibit transcription of the cytokine IL-1
Baldassare et al., J. Immunol. 1999 May 1;162(9):5367-73
Rapid Amplification of cDNA Ends (RACE)
RACE is a procedure for amplification of nucleic acid sequences from a messenger
RNA template between a defined internal site and unknown sequences at either the
3' or the 5' -end of the mRNA
2 Types of RACE: 5′ RACE and 3′ RACE
Why would you use RACE?
Amplify and characterize regions of unknown sequences
-oramplification of rare messages for which little sequence information is known
Detailed Information can be found at:
http://www.invitrogen.com/content/sfs/manuals/5prime_race_man.pdf
3′ RACE
5′ RACE
Anneal a Gene Specific
Primer (GSP1) to mRNA
Anneal a oligo dT Primer (with anchor sequence) to mRNA
mRNA
5′
AAAAAA 3′
GSP1
mRNA 5′
AAAAAA 3′
TTTTTT
Copy mRNA to cDNA using
reverse transcriptase and GSP1
- 5′
Copy mRNA to cDNA using reverse transcriptase
and oligo dT Primer+Anchor Sequence
mRNA 5′
cDNA 3′
5′
AAAAAA 3′
Degrade mRNA with RNase
mRNA 5′
cDNA 3′
AAAAAA 3′
TTTTTT
- 5′
cDNA
Degrade mRNA with RNase H
5′
3′
Treat cDNA and with
TdT and dCTP and Purify
TTTTTT
cDNA 3′
- 5′
3′- CCCCCC
Perform PCR using Gene Specific Primer (GSP1)
And Anchor Primer Complement (APC1)
PCR is performed using nested
GSP2 and Anchor Primer
 Companies offer special/custom
Anchor Primers
GSP1
cDNA 3′
TTTTTT
- 5′
APC1
5′
5′- GGGGGG
3′- CCCCCC
5′
GSP2
3′ RACE
PCR product
5′ RACE
PCR product
Exon Trapping
Exon Trapping is used to isolate the transcribed sequences (exons) of a gene from genomic
DNA
The exon trapping methods and vector were developed Alan Buckler et al.
Basic Protocol:
1) Random segments of chromosomal DNA are inserted into an intron present within a
mammalian expression vector
2) The cloned DNA is transfected into COS-7 cells
3) Amplified exons are spliced such that the vector and genomic exons are paired
4) Cytoplasmic mRNA is harvested and screened by PCR amplification for the acquisition of
an exon from the genomic fragment  the presence of two BstX I restriction sites flanking
the MCS helps minimize the recovery of vector-vector splicing or cryptic splicing
Publication: Buckler_Orig_Paper.pdf (Biochemistry website)
More information can be found at:
http://www.invitrogen.com/content/sfs/manuals/18449017.pdf
and Online Supplement
Splicing
 consensus sequences
3 splice site
splice acceptor site
5 splice site
splice donor site
intron
exon intron
AG GU AAGU
G
MCS
exon
NCAG G
mammalian expression vector
DS
exon intron
intron
AS
DS
AS
exon
intron
AG GU AAGU
G
intron
exon
NCAG G
Genomic DNA
containing an exon
flanked by introns
G
AG
exon
exon
exon
Microarray Technology
A technique scientist use to allow them to easily detect and measure the expression of
thousands of genes at one time.
Involves a DNA glass slide that is fixed with tiny amounts of a large number of singlestranded DNA fragments.
Uses:
• Studying differences in gene expression amongst a variety of genes in one organism
• Studying differences in gene expression between genetically similar organisms
• Compare cancerous tissue with noncancerous tissue
General Protocol:
1) Hybridization: Make labeled cDNA from mRNA and apply to the DNA chip
2) Rinse off excess cDNA and scan for fluorescence
3) Each fluorescent spot will indicate that the cDNA strand was complimentary to the strand
on the DNA chip
4) Ratio of fluorescence emission indicates relative abundance of each mRNA
Interesting articles on the Biochemistry website:
EricLander_Microarray.pdf
Brown_Botstein_Microarray.pdf
END