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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 l.jpg
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 l.jpg

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’


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InitialDNA

1

2

4

8

Number of DNA molecules



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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.


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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


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  • 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.


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How do we accurately quantify the amount of DNA? analyzed by agarose gel electrophoresis.

Real-time PCR


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Real Time PCR analyzed by agarose gel electrophoresis.


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3. intensifier analyzed by agarose gel electrophoresis.

5. ccd detector 350,000 pixels

1. halogen tungsten lamp

2b. emission filters

2a. excitation filters

4. sample plate


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Level off/ plateau analyzed by agarose gel electrophoresis.

Log phase

2

4

8

16

32

Amplification Plot of real-time PCR

DNA copy number (log)

PCR cycle (Ct)


Dna sequencing l.jpg
DNA sequencing analyzed by agarose gel electrophoresis.

  • 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 l.jpg
Sequence analysis analyzed by agarose gel electrophoresis.

  • 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.


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A C G T analyzed by agarose gel electrophoresis.

  • 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


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This is great but… analyzed by agarose gel electrophoresis.

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 l.jpg
07_03.jpg analyzed by agarose gel electrophoresis.


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Beckman CEQ 2000, 8 capillary electrophoresis and detected by a flourometer.

ABI Prism 3730, 96 capillary


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Affymetrix Gene Chip electrophoresis and detected by a flourometer.


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Affymetrix Expression Arrays electrophoresis and detected by a flourometer.

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


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Analysis of microarrays electrophoresis and detected by a flourometer.

  • 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.


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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 l.jpg
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 l.jpg
Data partitioning clinically important: cancerPatient 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.


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Another microarray example: understanding malaria cancer

IDC Transcriptomes for three P.falciparum strains

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


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Yeast 2 Hybrid cancer

Allows the geneticdetection of

physical interactions between

proteins


Yeast two hybrid assay l.jpg
Yeast Two Hybrid Assay cancer

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).


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GAL4 has two domains: cancer

one binds DNA (UAS) ,

the other activates transcription .

GAL1

{

UAS

GAL4

{

{

DNA-binding

Activating

GAL1

GAL1

POL

POL


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If protein X and Protein Y interact, cancer

they lead to expression of the

reporter gene.


Two hybrid assay l.jpg
Two-hybrid assay cancer

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


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Two-Hybrid cancerSelection

UAS-Reporter

GAL4-dbd/BAIT

GAL4-ad/X

Library

No expression

UAS-Reporter

GAL4-dbd/BAIT

GAL4-ad/X

Expression