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