• Polymerase chain reaction:

Starting with VERY SMALL AMOUNTS OF DNA (sometimes a few molecules), one can amplify the DNA enough to detect it by electrophoresis.

Polymerase Chain Reaction (PCR)

5’3’

5’3’

5’ 3’

5’ 3’

5’

5’

3’

3’

5’ 3’

5’3’

5’3’

5’3’

5’3’5’ 3’

5’ 3’

5’ 3’

5’3’

5’ 3’

5’3’5’ 3’

5’5’ 3’3’

5’

5’3’

5’ 3’

5’ 3’

3’

5’3’

5’ 3’

5’ 3’

5’3’

Denaturation

Annealing

Extension

Cycle 1

Cycle 2

Cycle 3

• Rate of PCR 2n

InitialDNA

8421Number of DNA molecules

CYCLE NUMBER DNA copy number0 11 22 43 84 165 326 647 1288 2569 512

10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000

Copies of DNA=2N

0.0E+00

2.0E+08

4.0E+08

6.0E+08

8.0E+08

1.0E+09

1.2E+09

1.4E+09

1.6E+09

1.8E+09

0 5 10 15 20 25 30 35

PCR cycle

RT-PCR• Polymerase chain reaction amplification of cDNA

can also be used to detect specific transcripts in a RNA sample.

• In this procedure, known as RT-PCR, reverse transcriptase is used to copy all of the mRNAs in an RNA sample into cDNA.

• Usually, oligo dT molecules, that anneal to the poly A tails of the mRNA, are used as primers.

• This single stranded cDNA can then be amplified by PCR using primers that anneal to a specific transcript sequence.

RT-PCR

AAA(A)n

5‘-CapmRNA

(dT)12~18 primer anneal

AAA(A)n

3‘ 5‘

Reverse transcriptasedNTP

5‘

cDNA:mRNA hybridRegular

PCRAAA(A)n

• The amplified DNA fragments that are produced can by analyzed by agarose gel electrophoresis.

• The amount of amplified fragment produced is proportional to the amount of target mRNA in the original RNA sample.

• Although less quantitative than Northern blots, RT-PCR is extremely sensitive and can be used to detect very rare mRNA species.

How do we accurately quantify the amount of DNA?

Real-time PCR

Real Time PCR

2a. excitation filters

2b. emission filters

1. halogen tungsten lamp

4. sample plate

3. intensifier

5. ccd detector 350,000 pixels

Log phase Level off/ plateau

32

16

8

4

2

Amplification Plot of real-time PCRD

NA

cop

y nu

mbe

r (lo

g)

PCR cycle (Ct)

DNA sequencing

• The Sanger di-deoxy method involves the synthesis of DNA by a DNA polymerase.

• DNA synthesis is terminated at specific nucleotides by the incorporation of di-deoxy nucleotides that are missing the 3’ OH.

• Sequencing is used to determine the precise order of nucleotides in a DNA molecule.

Sequence analysis

• Four different reactions produce DNA fragments that are terminated (randomly) at each of the four nucleotides.

• These samples are resolved by electrophoresis.

• The shortest fragments, those terminated closest to the primer, run faster than the longer fragments.

A C G T

• DNA sequencing reactions can be radioactively labeled.

• Bands detected by X-ray film exposure.

• Sequence can be read in the 5’ to 3’ direction from the bottom of the image towards the top.

GCAAT

A

CTA

This is great but…

Wouldn’t it be great to run everything in one lane?Saves space and time, more efficient

Fluorescently label the ddNTPs so that they each have a different color…

07_03.jpg

• Automated DNA sequencers use dideoxy terminators labeled with four different fluorescent dyes.

• All four reactions can be carried out simultaneously in a single reaction.

• Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer.

• The DNA sequence is read automatically.

Beckman CEQ 2000, 8 capillary ABI Prism 3730, 96 capillary

Affymetrix Gene Chip

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are needed to see this picture.

Affymetrix Expression Arrays

http://www.affymetrix.com/technology/ge_analysis/index.affx

Analysis of microarrays

• Microarrays allow for the simultaneous analysis of the expression of thousands of mRNAs.

• Useful for determining changes in gene expression patterns from one sample tissue to another.

• For example, microarrays have been used to study differences in gene expression in different tumor tissues.

Genes with similar expression patterns are clustered together.

Gene expression patterns can be associated with different disease patterns.

Microarray example #1: Biomarker identification - lung cancer

SamplesSamples

Gen

esG

enes

Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.

60

Cum

. Sur

viva

l

Time (months)

0

.2

.4

.6

.8

1

0 10 20 30 40 50

Cum. Survival (Group 3)

Cum. Survival (Group 2)

Cum. Survival (Group 1)

p = 0.002for Gr. 1 vs. Gr. 3

Data partitioning clinically important: Patient survival for lung cancer subgroups

Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.

Llinas, M. et al. Nucl. Acids Res. 2006 34:1166-1173; doi:10.1093/nar/gkj517

IDC Transcriptomes for three P.falciparum strains

Another microarray example: understanding malaria

Yeast 2 Hybrid

Allows the genetic detection of physical interactions between

proteins

Yeast Two Hybrid Assay• The two-hybrid system is a molecular genetic tool which

facilitates the study of protein-protein interactions. • If two proteins interact, then a reporter gene is

transcriptionally activated. – e.g. gal1-lacZ - the beta-galactosidase gene

• A colour reaction can be seen on specific media. • You can use this to

– Study the interaction between two proteins which you expect to interact

– Find proteins (prey) which interact with a protein you have already (bait).

GAL4 has two domains:one binds DNA (UAS) ,the other activates transcription .

GAL1

{{

GAL4

DNA-bindingDNA-binding Activating

GAL1

POL

GAL1

POL

{

UAS

If protein X and Protein Y interact,they lead to expression of the reporter gene.

Two-hybrid assay

1.

2. 3.

4.

AB

Fields S. Song O.Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163

UASG

GAL4-DBD

SNF1

SNF4

Transcription activation domain

Allows growth on galactose

GAL1

Two-Hybrid Two-Hybrid SelectionSelectionUAS-Reporter

GAL4-dbd/BAIT

GAL4-adGAL4-ad/X/XLibraryLibrary

UAS-Reporter

GAL4-dbd/BAIT

GAL4-ad/X

ExpressionExpression

No expressionNo expression