Application of PCR and Microarray in Molecular Biology

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Application of PCR and Microarray in Molecular
Biology
Michael Chan, PhD Postdoctoral
Researcher Integrated Cancer Biology Program
, Comprehensive Cancer Center The Ohio State
University
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Animal Cell
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DNA and Chromatin
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The Central Dogma of Molecular Biology
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Control of gene expression
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Measuring Gene Expression
Idea measure the amount of mRNA to see which
genes are being expressed in (used by) the cell.
Measuring protein might be more direct, but is
harder.
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Retrovirus
Reverse transcriptase makes a complementary DNA
copy from RNA. (Howard M. Temin/David Baltimore,
1975 Nobel Prize in Physiology or Medicine)
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(Reverse Transcription)
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Reverse transcription
cDNA strands, complementary to the mRNA
G U A A U C C U C
mRNA
Reverse transcriptase
T T A G G A G
cDNA
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Polymerase Chain Reaction (PCR)
Thermus aquaticus, a thermophilic bacteria
discovered in 1969 in hot spring of Yellowstone
National park . It can tolerate high temperature.
The DNA polymerase (Taq polymerase) was isolated.
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Polymerase Chain Reaction (PCR)
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Gel Electrophoresis
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Copies of DNA2N
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How do we accurately quantify the amount of DNA?
Real-time PCR
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Real Time PCR
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3. intensifier
5. ccd detector 350,000 pixels
1. halogen tungsten lamp
2b. emission filters
2a. excitation filters
4. sample plate
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Amplification Plot of real-time PCR
DNA copy number (log)
PCR cycle (Ct)
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Application
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Journal of Virological Methods 102119-128, 2002
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Journal of Clinical Virology 28233-238, 2003
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What if 10,000 genes are to be quantitated by
PCR??
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Expression Microarray

• High throughput technology that allows detection
  of thousands of gene simultaneously
• Apply hybridization principle (imagine thousand
  of Southern blots)
• Data require further statistical analysis
  (bioinformatics)

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Southern Blot Analysis
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cDNA Microarray analysis
Adapted from Bryant et al., 2004, Lancet
infectious disease
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Biological question Differentially expressed
genes Sample class prediction etc.
Experimental design
Microarray experiment
Image analysis
Normalization
Estimation
Testing
Clustering
Discrimination
Biological verification and interpretation
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Principal Uses of Microarrays

• Genome-scale gene expression analysis
• Differential gene expression between two (or
  more) sample types
• Responses to environmental factors
• Disease processes (e.g. cancer)
• Effects of drugs
• Identification of genes associated with clinical
  outcomes (e.g. survival)

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Ralph et al., Mol Ecol 151275-1297, 2006
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Clinical Cancer Research 112625-2636, 2005
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Limitation of expressing microarray

• Detecting mRNA level not protein level
• Many factors can affect microarray result
• Chip type
• sample preparation
• data analysis
• Require bioinformatics for data analysis.

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Other types of microarray platform

• ChIP (Chromatin Immunoprecipitation) microarray
• Methylation microarray (Differential Methylation
  Hybridization)
• CGH (comparative genomic hybridization) microarray

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The End