Transcript Microarray Analysis


Genomic Analysis
Chapter 19
Overview of topics to be discussed

How to sequence genomic DNA
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(we will have to touch briefly on polymerase chain reaction—a topic
from the upcoming “PCR” lab).
cDNA
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Microarray Analysis
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The Human Genome Project
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How to use information from any genome sequence:

Single Nucleotide Polymorphisms

Predicting Function from Sequence
 cDNA:
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
mRNA
Reverse transcriptase
cDNA can also be studiedwww.bio.davidson.edu/Courses/genomics/RTPCR/RT_PCR.html :
SEQUENCING DNA
Sanger-Dideoxymethod
Dideoxy key point!
Sanger
Method dideoxynucleotides: lack a 3’ OH
But
have the 2’ and 3’ have hydrogen
NO
DNA EXTENSION
DNA PCR amplification is performed prior to sequence analysis
Autoradiogram
Can you sequence without using radioactivity?
Radioactive!
Yes: Example of Labelled Probes
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ddGTP
ddATP
ddTTP
ddCTP
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Orange
Black
Red
Blue
Microarray Analysis
Microarray Protocol

DNA or cDNA is added
robotically to chip
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Chips exposed to labeled cDNA
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Label monitored by laser
Example of: Microarrays from breast tumors

cDNA from breast tumors from 2 groups of
patients
Metastatic Cancer
Disease Free
Microarrays
of
70
genes
(row is a patient; column a gene)
Metastatic
cancers
Genomic DNA
Transcriptome
DATA BASE
Proteome
Human Genome Project
June, 2002
Dr. Craig Venter
President Clinton
Dr. Francis Collins
The initial draft of the Human Genome
•3.4 billion base pairs
•32,000 genes
•Average gene 27,000 bases
•Chromosome 17, 19, 22 high density
•May encode 96,000 proteins
Mouse: Mus musculus

2.6 billion bp

99% in common with human genome
Genomic Projects: provide us
information about:

Expressed-sequence tags (ESTs) Need to understand cDNA

Single-nucleotide polymorphisms (SNPs) & Haplotypes
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Variable Number Tandem Repeats (VNTRs)
Haplotype: A set of SNPs and other variants on the same chromosome
Variable number tandem repeats
(VNTRs) in multiple loci
Short stretches of DNA base pairs that are repeated
at multiple loci in the chromosomes.
GAGGAG
GAGGAGGAG
GAGGAGGAG
GAGGAGGAGGAGGAG
GAGGAGGAGGAG
GAG
GAGGAGGAG
GAGGAGGAGGAG
GAGGAGGAGGAGGAGGAG
GAGGAGGAGGAGGAGGAG
Predicting Function from
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Homology Search
Computational method prediction
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Paralog

Ortholog
Paralogs: Homologous sequences in same
species
Orthologs
Homologous
Sequences in
Different species