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Polymerase Chain Reaction (PCR) 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. 5’ 3’ 3’ 5’ Denaturation 5’ 5’ 3’ 3’ 3’ 5’ Annealing Cycle 1 3’ 5’ Extension 5’ 3’ 5’

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polymerase chain reaction pcr
Polymerase Chain Reaction (PCR)
  • 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.

slide2

5’

3’

3’

5’

Denaturation

5’

5’

3’

3’

3’

5’

Annealing

Cycle 1

3’

5’

Extension

5’

3’

5’

5’

5’

5’

5’

5’

5’

5’

3’

3’

3’

3’

3’

3’

3’

3’

3’

5’

5’

3’

3’

3’

3’

3’

3’

3’

3’

3’

5’

5’

5’

5’

5’

5’

5’

5’

Cycle 2

3’

5’

5’

3’

Cycle 3

3’

5’

slide3
Rate of PCR 2n

InitialDNA

1

2

4

8

Number of DNA molecules

rt pcr
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.
slide6

AAA(A)n

AAA(A)n

RT-PCR

5‘-Cap

mRNA

AAA(A)n

(dT)12~18

primer

anneal

3‘

5‘

Reverse transcriptase

dNTP

5‘

Regular

PCR

cDNA:mRNA hybrid

slide7
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.
slide11

3. intensifier

5. ccd detector 350,000 pixels

1. halogen tungsten lamp

2b. emission filters

2a. excitation filters

4. sample plate

slide12

Level off/ plateau

Log phase

2

4

8

16

32

Amplification Plot of real-time PCR

DNA copy number (log)

PCR cycle (Ct)

dna sequencing
DNA sequencing
  • Sequencing is used to determine the precise order of nucleotides in a DNA molecule.
  • 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.
sequence analysis
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.
slide25

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.

A

A

T

C

T

A

A

C

G

slide26

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…

slide28

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

  • All four reactions can be carried out simultaneously in a single reaction.
slide29
Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer.
  • The DNA sequence is read automatically.
slide30

Beckman CEQ 2000, 8 capillary

ABI Prism 3730, 96 capillary

affymetrix expression arrays
Affymetrix Expression Arrays

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

analysis of microarrays
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.
slide36

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
Microarray example #1: Biomarker identification - lung cancer

Samples

Genes

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

data partitioning clinically important patient survival for lung cancer subgroups
Data partitioning clinically important: Patient survival for lung cancer subgroups

1

Cum. Survival (Group 1)

.8

Cum. Survival (Group 2)

Cum. Survival (Group 3)

.6

Cum. Survival

.4

.2

0

0

10

20

30

40

50

60

Time (months)

p = 0.002

for Gr. 1 vs. Gr. 3

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

slide39

Another microarray example: understanding malaria

IDC Transcriptomes for three P.falciparum strains

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

slide40

Yeast 2 Hybrid

Allows the geneticdetection of

physical interactions between

proteins

yeast two hybrid assay
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).

slide42

GAL4 has two domains:

one binds DNA (UAS) ,

the other activates transcription .

GAL1

{

UAS

GAL4

{

{

DNA-binding

Activating

GAL1

GAL1

POL

POL

slide43

If protein X and Protein Y interact,

they lead to expression of the

reporter gene.

two hybrid assay
Two-hybrid assay

SNF4

1.

B

SNF1

A

3.

2.

GAL4-DBD

Transcription activation domain

UASG

4.

Fields S. Song O.

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

GAL1

Allows growth on galactose

slide45

Two-Hybrid Selection

UAS-Reporter

GAL4-dbd/BAIT

GAL4-ad/X

Library

No expression

UAS-Reporter

GAL4-dbd/BAIT

GAL4-ad/X

Expression