Polymerase Chain Reaction and DNA Sequencing · PPT file · Web view ·...

• 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)

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Denaturation

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• 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

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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.

RT-PCR

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(dT)12~18 primer anneal

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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.

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

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Amplification Plot of real-time PCRD

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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.

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• 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

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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…

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• 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

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