Statistics for Microarrays

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Statistics for Microarrays
Biological background Gene Expression and
Molecular Laboratory Techniques
Class web site http//statwww.epfl.ch/davison/te
aching/Microarrays/
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Basic principles in physics, chemistry and biology
Principles Known?
Physics Matter
Chemistry Compound
Biology Organism
Elementary Particles Yes
Genes No
Elements Yes
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Central Paradigm
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(RT)
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Protein Synthesis
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Transcription

• Transcription is a complex process involving
  several steps and many proteins (enzymes)
• RNA polymerase synthesizes a single strand of RNA
  against the DNA template strand (anti-sense
  strand), adding nucleotides to the 3 end of the
  RNA chain
• Initiation is regulated by transcription factors,
  including promoters, usually an initiator element
  and TATA box, usually lying just upstream (at the
  5 end) of the coding region
• 3 end cleaved at AAUAAA, poly-A tail added

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Exons and Introns

• Most of the genome consists of non-coding regions
• Some non-coding regions (centromeres and
  telomeres) may have specific chomosomal functions
• Other non-coding regions have regulatory purposes
• Non-coding, non-functional DNA often called junk
  DNA, but may have some effect on biological
  functions
• The terms exon and intron refer to coding and
  non-coding DNA, respectively

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Intron Splicing
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Transcription Overview
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Transcription Illustration
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Translation

• The AUG start codon is recognized by
  methionyl-tRNAiMet
• Once the start codon has been identified, the
  ribosome incorporates amino acids into a
  polypeptide chain
• RNA is decoded by tRNA (transfer RNA) molecules,
  which each transport specific amino acids to the
  growing chain
• Translation ends when a stop codon (UAA, UAG,
  UGA) is reached

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Translation Illustrated
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From Primary Transcript to Protein
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Alternative Splicing (of Exons)

• How is it possible that there are over 1,000,000
  human antibodies when there are only about 30,000
  genes?
• Alternative splicing refers to the different ways
  the exons of a gene may be combined, producing
  different forms of proteins within the same
  gene-coding region
• Alternative pre-mRNA splicing is an important
  mechanism for regulating gene expression in
  higher eukaryotes

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Molecular Laboratory Techniques

• Hybridizing DNA
• Copying DNA
• Cutting DNA
• Probing DNA

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Hybridization

• Hybridization exploits a potent feature of the
  DNA duplex the sequence complementarity of the
  two strands
• Remarkably, DNA can reassemble with perfect
  fidelity from separated strands
• Strands can be separated (denatured) by heating

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Polymerase Chain Reaction (PCR)

• PCR is used to amplify (copy) specific DNA
  sequences in a complex mixture when the ends of
  the sequence are known
• Source DNA is denatured into single strands
• Two synthetic oligonucleotides complementary to
  the 3 ends of the segment of interest are added
  in great excess to the denatured DNA, then the
  temperature is lowered
• The genomic DNA remains denatured, because the
  complementary strands are at too low a
  concentration to encounter each other during the
  period of incubation, but the specific
  oligonucleotides hybridize with their
  complementary sequences in the genomic DNA

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PCR, ctd

• The hybridized oligos then serve as primers for
  DNA synthesis, which begins upon addition of a
  supply of nucleotides and a temperature resistant
  polymerase such as Taq polymerase, from Thermus
  aquaticus (a bacterium that lives in hot springs)
• Taq polymerase extends the primers at
  temperatures up to 72C
• When synthesis is complete, the whole mixture is
  heated further (to 95C) to melt the newly formed
  duplexes
• Repeated cycles (2530) of synthesis (cooling)
  and melting (heating) quickly provide many DNA
  copies

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Types of Viruses
A virus is a nucleic acid in a protein coat.
Reverse transcriptase makes a complementary DNA
copy from RNA.
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Reverse transcription
Clone 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
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
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RT-PCR
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Restriction Enzymes Cut DNA
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Restriction Enzymes

• When a bacterium is invaded by a DNA-containing
  organism (e.g. virus), it can defend itself with
  restriction enzymes (REs also called restriction
  endonucleases)
• REs recognize a specific short sequence of DNA
  and cut both strands
• The recognition sequence is typically a
  palindrome i.e. the sequence in one strand is
  the same as in the other, read in the other
  direction (e.g. GAATTC)
• REs named after the bacteria in which they occur,
  plus sequence number (e.g. Eco RI)

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RE Example (Eco RI)
(cut) 5 GAATTC 3 3
CTTAAG 5 (cut)
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Probing DNA

• One way to study a specific DNA fragment within a
  genome is to probe for the sequence of the
  fragment
• A probe is a labeled (usually radioactive or
  fluorescent) single-stranded oligonucleotide,
  synthesized to be complementary to the sequence
  of interest probe sequence is known
• Attach single-stranded DNA to a membrane (or
  other solid support) and incubate with the probe
  so that it hybridizes
• Visualize the probe (e.g. by X-ray for
  radioactive probes)

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The Southern blotting technique
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Sample Autoradiogragh (Gel)
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Types of Blots

• Southern Blot use DNA to probe DNA
• Northern Blot use DNA to probe RNA
• Western Blot use antibodies to probe Protein

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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 would be more direct, but is
currently harder.
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Microarrays provide a means to measure gene
expression
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Areas Being Studied with Microarrays

• Differential gene expression between two (or
  more) sample types
• Similar gene expression across treatments
• Tumor sub-class identification using gene
  expression profiles
• Classification of malignancies into known classes
• Identification of marker genes that
  characterize different tumor classes
• Identification of genes associated with clinical
  outcomes (e.g. survival)

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cDNA microarray experiments

• mRNA levels compared in many different contexts
• Different tissues, same organism (brain v.
  liver)
• Same tissue, same organism (ttt v. ctl, tumor v.
  non-tumor)
• Same tissue, different organisms (wt v. ko, tg,
  or mutant)
• Time course experiments (effect of ttt,
  development)
• Other special designs (e.g. to detect spatial
  patterns).

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Web animation of a cDNA microarray experiment
http//www.bio.davidson.edu/courses/genomics/chip/
chip.html
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Yeast genome on a chip
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Brief outline of steps for producing a microarray

• cDNA probes attached or synthesized to solid
  support
• Hybridize targets
• Scan array

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cDNA microarrays
cDNA clones
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cDNA microarrays

• Compare the genetic expression in two samples of
  cells

PRINT cDNA from one gene on each spot
SAMPLES cDNA labelled red/green
e.g. treatment / control normal / tumor
tissue
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HYBRIDIZE Add equal amounts of labelled cDNA
samples to microarray.
SCAN
Laser
Detector
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Quantification of expression

• For each spot on the slide we calculate
• Red intensity Rfg - Rbg
• (fg foreground, bg background) and
• Green intensity Gfg - Gbg
• and combine them in the log (base 2) ratio
• Log2( Red intensity / Green intensity)

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Gene Expression Data

• On p genes for n slides p is O(10,000), n is
  O(10-100), but growing,

Slides
slide 1 slide 2 slide 3 slide 4 slide 5 1
0.46 0.30 0.80 1.51 0.90 ... 2 -0.10 0.49
0.24 0.06 0.46 ... 3 0.15 0.74 0.04 0.10
0.20 ... 4 -0.45 -1.03 -0.79 -0.56 -0.32 ... 5 -0.
06 1.06 1.35 1.09 -1.09 ...
Genes
Gene expression level of gene 5 in slide 4

Log2( Red intensity / Green intensity)
These values are conventionally displayed on a
red (gt0) yellow (0) green (lt0) scale.
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Biological question Differentially expressed
genes Sample class prediction etc.
Experimental design
Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg), (Gfg, Gbg)
Normalization
R, G
Estimation
Testing
Clustering
Discrimination
Biological verification and interpretation