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04. PCR
in vitro enzymatic amplification of DNA
- From Kleppe & Khorana to Kary Mullis
=> from ‘repair synthesis’ to ‘exponential amplification’ via synthetic oligonucleotides
- basic concept of amplification: “when product becomes substrate”
the PCR cycle: denaturation, annealing, elongation: (2n - 2n) amplicon molecules
- properties of Taq polymerase:
- single polypeptide chain of 94 kDa; 200,000 U/mg
- Topt = 70 - 80 °C
- Kcat : up to 150 nt per second per enzyme molecule
(at 55°C still about 24 nt/s)
- no 3' => 5' exo activity, but has 5' => 3' exo activity
(=> misincorporation frequencies 10-5 to 2x10-4 per nt per cycle)
- terminal transferase activity : adds an extra A (or other nt) at 3' ends
- large diversity of templates, only minimal amounts required
in situ lysis of E.coli, B.subtilis, Streptomyces, yeasts, animals cells, plant
protoplasts may be sufficient; fossile material, microscope slides, blood stain, etc.
- problem of sensitivity versus risk of contamination
- size range of amplicon: 200-2000 bp versus 2 - 5 kb versus 5 - 50 kb (long-range PCR)
- amplification process:
temperature profile, ‘touch-down’ PCR,
hot-start procedure (temperature or antibodies)
A = E(n).(2n-2n)
amplification
Nf = No (1 + E(n))n
number of molecules
Em = (Nf/No)1/n -1
mean efficieny per cycle
- decreasing efficiency at 1012 "targets" (or even less)
- increasing amplicon length : "long-range" PCR :
(about 1 g of a 1 kb DNA fragment)
- go across defects
- use mixture of polymerases
- primer choice: - size, %G+C (40-60%, balanced between both primers)
- avoid complementarity between primer ends, and within primer
- mismatches, tails
- Other factors: buffer, additives (KCl, DMSO, ...), Mg2+ concentration, …
G. Volckaert
PCR
12/02/2016
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- Basic goals:
- analytical (identify presence of sequence) (in particular if only minute amounts present)
- synthetic (increase amount of DNA fragment, make new combinations, etc...)
examples of application
- clone analysis (replacing plasmid isolation + restriction analysis)
- screening: genetic errors (diploid => homozygote, heterozygote) (including prenatal)
- screening for viruses and other pathogens (e.g. HIV, HCV, etc.)
- diagnosis of bacterial infections: medical, veterinarian, food industry
- Variations: - nested PCR
- RT-PCR (use of reverse transcriptase, use of Tth polymerase)
- A-PCR : asymmetric PCR
- inverse PCR, RAcE (see Chapter: DNA libraries)
- SOE (see also Chapter: Directed mutagenesis)
- incorporation of nucleotide analogs and tagging (see above)
- (semi-)quantitative PCR, competitive PCR, real-time PCR
- LIC : 'ligation independent cloning' (not a PCR technique, but fragments to be cloned
ordinarily prepared by PCR)
Addendum
- LCR: technique of cyclic ligase reaction (with thermoresistant ligase) for use in diagnosis (e.g. of mutants)
=> amplification of ligated oligonucleotides
applicable only with ligases devoid of blunt-end ligation activity
G. Volckaert
PCR
12/02/2016