Primer-mediated enzymatic

amplification of DNA sequences

Definition:

8 PCR

PCR (Polymerase Chain Reaction)

Kary B. Mullis

Nobel price in chemistry in 1993

* 1944

R Saiki (1985) Science 230: 1350

8.1 Principle of the PCR Reaction

Thermostable DNA Polymerases

Taq polymerase

8.2 Cloning of PCR Fragments

Addition of restriction sites

T-A overhang cloning

Generation of sticky-end PCR products

Splicing by extension overlap (SOE)

Seamless cloning

Addition of Restriction Sites

The T-A Overhang Cloning

TA Holton (1991) Nucleic Acids Res. 19: 1156 D Marchuk (1991) Nucleic Acids Res. 19: 1154

Disadvantages:1. No orientation-specific

cloning 2. No proof-reading with Taq

Generation of Sticky-End Products

A Walker (2008) Plasmid 59: 155

Advantage: No restriction enzyme treatment necessary

Splicing by Overlap Extension (SOE) PCR

A.N. Warrens (1997) Gene 186: 29

⇓ ⇓

Promoter Gene

Overlap: 15-20 nucleotides

⇓

8.3 Specific PCR Reactions

RT – PCRRACE – PCR Inverse PCR PCR for strain and species identificationMultiplex PCR Real Time PCR

RT-PCR (Reverse Transcription)

Applications: 1. Detection and quantification of transcripts

present in low amounts2. Cloning of genes

Reverse Transcription PCR: Detection and Quantization of

Transcripts

Labeled cDNA

Reverse Transcription

PCR: Cloning of Genes

RACE-PCR (Rapid Amplification

of cDNA Ends)

Problem:The 5' end or the 3' end or both ends of a eukaryotic gene is (are) missing

Known: At least the central part of the gene

Additional use: Quantization of transcripts

3' RACE

1. Preparation of total RNA

2. Addition of Oligo(dT) primer with restriction site (= anchor primer)

3. Internal sense primer 4. Amplification

5' RACE

1. Preparation of total RNA

2. Internal antisenseprimer

3. A-tailing 4. Oligo(dT) primer

with restriction site

5. PCR

Inverse PCR

Goal:To first amplify and then determine the DNA sequences on both sides of a known sequence

The Principle of Inverse PCR

ligate inverse PCR

RAPD PCR (Randomly Amplified

Polymorphic DNA)

Objective:

To study the phylogenetic relationship of strains

RAPD Primer with an Arbitrary Sequence

12-mer

First step:Low stringency

Second step:High stringency

Linear PCR

Genomic Fingerprints of Rice and Streptococcus Strains

Multiplex PCR

Definition:

Use of multiple primer pairs in the same PCR reaction

Multiplex PCR Using Nine Different Primer Pairs

Respiratory diseases of the pig

Real Time PCR

Definition: Real time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to endpoint detection

Real Time PCR Assays

1. The amount of amplicons is measured after each PCR cycle by the increase of a fluorescence dye

2. Available instruments use three fluorescencemethods to monitor amplicon production:* TaqMan probes * FRET probes using the LightCycler * Molecular Beacons * SYBER Green

Principle of TaqMan Probes

1. The TaqMan probe is shown annealed to the target DNA; contains a reporter and quencher dye

Principle of TaqMan Probes

2. During the PCR reaction, a complementary strand of DNA is synthesized and the 5' exonuclease activity of Taq DNA polymerase excises the reporter dye

3. Fluorescence of the reporter dye occurs as a result of separation of the reporter dye from the quencher dye

Principles of FRET (FluorescentResonance Energy Transfer) Using

the LightCycler

Two probes hybridize to the DNA separated by a short distance (1 - 5 n) This allows energy transfer from the donor to the acceptor dye The more molecules, the higher the FRET

Principle of Molecular Beacon Probes

1. The molecular beacon probe has a hairpin form complementary to the probe where the stem hybrid keeps the fluorophore (reporter dye) close to the quencher dye

2. Upon annealing the reporter dye is separated from the quencher restoring fluorescence

Application of Molecular Beacons

1. To monitor the amplification of DNA during real-time PCR

2. To identify Single Nucleotide Polymorphisms (SNP)

3. To detect pathogens4. To quantify gene expression

M Rajendran (2003) Nucleic Acids Res. 31: 5700

SYBER Green Method

Principle:SYBER Green intercalates into dsDNA, but not ssDNADisadvantage:Binds also to non-specific dsDNA products