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Statistics For Microarrays: Biological Background: Gene Expression and Molecular Laboratory Techniques

This document provides an overview of gene expression and molecular laboratory techniques used for microarrays. It discusses the processes of transcription and translation that lead to protein synthesis from DNA. Key techniques covered include PCR to amplify DNA, restriction enzymes that cut DNA at specific sequences, probes used to detect DNA fragments, and microarrays that allow comparing gene expression levels across many samples to study processes like differential expression between cell types. Microarrays provide a way to measure mRNA levels as a proxy for which genes are actively used in a cell.

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Varun Dwivedi
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22 views

Statistics For Microarrays: Biological Background: Gene Expression and Molecular Laboratory Techniques

This document provides an overview of gene expression and molecular laboratory techniques used for microarrays. It discusses the processes of transcription and translation that lead to protein synthesis from DNA. Key techniques covered include PCR to amplify DNA, restriction enzymes that cut DNA at specific sequences, probes used to detect DNA fragments, and microarrays that allow comparing gene expression levels across many samples to study processes like differential expression between cell types. Microarrays provide a way to measure mRNA levels as a proxy for which genes are actively used in a cell.

Uploaded by

Varun Dwivedi
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Statistics for Microarrays

Biological background: Gene Expression


and Molecular Laboratory Techniques

Class web site:


http://statwww.epfl.ch/davison/teaching/Microarrays/
Basic principles in physics, chemistry and biology

Principles Known?

Physics Chemistry Biology


Matter Compound Organism

Elementary Elements Genes


Particles

Yes Yes No
Central Paradigm
(RT)
Protein Synthesis
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
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
Intron Splicing
Transcription Overview
Transcription Illustration
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
Translation Illustrated
From Primary Transcript
to Protein
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
Molecular Laboratory Techniques

• Hybridizing DNA

• Copying DNA

• Cutting DNA

• Probing DNA
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
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
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
(BREAK)
Types of Viruses

A virus is a nucleic acid in a protein coat.


Reverse transcriptase makes a complementary
DNA copy from RNA.
Reverse transcription

Clone cDNA strands, complementary to the mRNA

mRNA GUAAUCCUC
Reverse
transcriptase

TT
AG
cDNA GA
G

CA
TTA G
GA
G AGGA
C ACTATTAAGG G G
CA CAT T
T
CCAAT
A G
A G
TT TG
GAAA
G
GGG
AGGG
AAGG
T T T G A
C A T T A G G AGG
CA
RT-PCR
Restriction Enzymes Cut DNA
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)
RE Example (Eco RI)

(cut)
5’ – GAATTC – 3’
3’ – CTTAAG – 5’
(cut)
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)
The Southern blotting technique
Sample Autoradiogragh (Gel)
Types of Blots

• Southern Blot – use DNA to probe DNA

• Northern Blot – use DNA to probe RNA

• Western Blot – use antibodies to probe

Protein
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.
Microarrays provide a means
to measure gene expression
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)
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).


Web animation of a cDNA microarray
experiment

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

• cDNA probes attached or synthesized to


solid support

• Hybridize targets

• Scan array
cDNA microarrays

cDNA clones
cDNA microarrays
Compare the genetic expression in two samples of cells

PRINT SAMPLES
cDNA from one cDNA labelled red/green
gene on each spot

e.g. treatment / control


normal / tumor tissue
HYBRIDIZE SCAN
Add equal amounts of Laser Detector
labelled cDNA samples to
microarray.
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)


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 ...
Genes 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 ...

Gene expression level of gene 5 in slide 4


= Log2( Red intensity / Green intensity)

These values are conventionally displayed


on a red (>0) yellow (0) green (<0) scale.
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

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