Biological background: Gene Expression and Molecular Laboratory Techniques Class web site: ...
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Biological background: Gene Expression and Molecular
Laboratory Techniques
Class web site: http://statwww.epfl.ch/davison/teaching/Microarrays/
Statistics for Microarrays
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Basic principles in physics, chemistry and biology
Principles Known?
Physics
Matter
Chemistry
Compound
Biology
Organism
ElementaryParticles
Yes
Elements
Yes
Genes
No
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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-tRNAi
Met
• 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 72˚C
• When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes
• Repeated cycles (25—30) of synthesis (cooling) and melting (heating) quickly provide many DNA copies
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(BREAK)
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Types of Viruses
Reverse transcriptase makes a complementary DNA copy from RNA.
A virus is a nucleic acid in a protein coat.
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Reverse transcription
Clone cDNA strands, complementary to the mRNA
G U A A U C C U C
Reverse transcriptase
mRNA
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
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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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.
Measuring Gene Expression
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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
PRINTcDNA from one gene on each spot
SAMPLEScDNA 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,
Genes
Slides
Gene expression level of gene 5 in slide 4
= Log2( Red intensity / Green intensity)
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 ...
These values are conventionally displayed on a red (>0) yellow (0) green (<0) scale.
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Biological questionDifferentially expressed genesSample class prediction etc.
Testing
Biological verification and interpretation
Microarray experiment
Estimation
Experimental design
Image analysis
Normalization
Clustering Discrimination
R, G
16-bit TIFF files
(Rfg, Rbg), (Gfg, Gbg)