PCR (Polymerase Chain Reaction) Primer-mediated enzymatic amplification of DNA sequences

Transcription

1 8 PCR PCR (Polymerase Chain Reaction) Definition: Primer-mediated enzymatic amplification of DNA sequences

2 Kary B. Mullis * 1944 R Saiki (1985) Science 230: 1350 Nobel price in chemistry in 1993

3 8.1 Principle of the PCR Reaction

4 Thermostable DNA Polymerases

5

6 Taq polymerase

7

8 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

9 Addition of Restriction Sites

10 The T-A Overhang Cloning Disadvantages: 1. No orientation-specific cloning 2. No proof-reading with Taq TA Holton (1991) Nucleic Acids Res. 19: 1156 D Marchuk (1991) Nucleic Acids Res. 19: 1154

11 Generation of Sticky-End Products Advantage: No restriction enzyme treatment necessary A Walker (2008) Plasmid 59: 155

12 Splicing by Overlap Extension (SOE) PCR Promoter Overlap: nucleotides Gene A.N. Warrens (1997) Gene 186: 29

13 8.3 Specific PCR Reactions RT PCR RACE PCR Inverse PCR PCR for strain and species identification Multiplex PCR Real Time PCR

14 RT-PCR (Reverse Transcription) Applications: 1. Detection and quantification of transcripts present in low amounts 2. Cloning of genes

15 Reverse Transcription PCR: Detection and Quantization of Transcripts Labeled cdna

16 Reverse Transcription PCR: Cloning of Genes

17 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

18 3' RACE 1. Preparation of total RNA 2. Addition of Oligo(dT) primer with restriction site (= anchor primer) 3. Internal sense primer 4. Amplification

19 5' RACE 1. Preparation of total RNA 2. Internal antisense primer 3. A-tailing 4. Oligo(dT) primer with restriction site 5. PCR

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

21 The Principle of Inverse PCR ligate inverse PCR

22 RAPD PCR (Randomly Amplified Polymorphic DNA) Objective: To study the phylogenetic relationship of strains

23 RAPD Primer with an Arbitrary Sequence 12-mer Linear PCR First step: Low stringency Second step: High stringency

24 Genomic Fingerprints of Rice and Streptococcus Strains

25 Multiplex PCR Definition: Use of multiple primer pairs in the same PCR reaction

26 Multiplex PCR Using Nine Different Primer Pairs Respiratory diseases of the pig

27 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

28

29 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 fluorescence methods to monitor amplicon production: * TaqMan probes * FRET probes using the LightCycler * Molecular Beacons * SYBER Green

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

31 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

32 Principles of FRET (Fluorescent Resonance 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

33 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

34 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 pathogens 4. To quantify gene expression M Rajendran (2003) Nucleic Acids Res. 31: 5700

35 SYBER Green Method Principle: SYBER Green intercalates into dsdna, but not ssdna Disadvantage: Binds also to non-specific dsdna products