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5. Enzymatical modification of DNA (including PCR)
Modifications at the nucleotide bases
methylations: restriction/modification systems : methylation at C5 or at A6 (NH2)
Dam & Dcm methylase (of E. coli) : GATC & CCWGG
CpG methylase
(methylation-dependent endonucleases in E. coli : McrA, McrBC, Mrr)
incorporations by PCR: e.g. biotin, digoxigenin
incorporation via modified dNTPs (often at C5 of thymine (uracil))
alternative: chemical modification in (one of) the primer
(see also Chemical DNA synthesis)
sequence-modification: incorporation of extra tail
Modifications at the termini : phosphate- or OH-group
removal of terminal phosphates
(alkaline) phosphatases (phosphomonoesterases: hydrolyse phosphomonoester bonds)
- removal from 5’, 3’, 2’ (wherever it is) ; leaves 5’-, 3’- or 2’-OH
- different properties, in particular in terms of stability (and inactivation)
- bacterial alkaline phosphatase (BAP): from E. coli
- calf intestinal alkaline phosphatase (CIAP)
- shrimp alkaline phosphatase (SAP)
hydrolysis of (terminal) multi-phosphates (a.o. mRNA CAP structure)
pyrophosphatase (cleaves phospho-anhydric bonds)
- cleaves CAP structure while leaving one phosphate group at the 5’-end
(since that is a phosphomonoester, not an anhydride)
- TAP : tobacco acid pyrophosphatase
addition of a (terminal) phosphate to a 5’-OH
T4-polynucleotide kinase
- transfers the -phosphate of ATP to the 5’-OH end
G. Volckaert
Enzymatical modification of DNA, including PCR
16/02/2016
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further tinkering at the termini
exonucleases : stepwise removal of monomers (or oligomers) from the ends
- exonuclease III, lambda exonuclease, exonuclease VII, T7 gene 6 exonuclease
- substrate specificity (ds versus ss), direction 3’=>5’ versus 5’=>3’, processivity, etc.
reactions with DNA-polymerases :
- (partial or complete) filling-in of 5’-protruding ends : dNTP substrates
- exonucleolytic activities
terminal deoxynucleotidyl transferase (TdT)
- template-independent addition of nucleotides at 3’-OH-ends (‘tailing’)
NTP, dNTP, ddNTP, cordicepin triphosphate
starter ss or ds, but at least NpNpN (and must be a deoxynucleotide strand)
poly(A)-polymerase : generation of a poly(A) tail at an RNA molecule (3’-OH)
Modification of sequence structural integrity of nucleic acid molecules
internal hydrolysis of the backbone chain: endonucleases (phospodiesterases)
- DNaseI (‘random’ nicking : Mg2+
ds-cleavage : Mn2+ )
- nuclease S1, mung bean nuclease : specificity for single strands
- RNases: RNase T1, RNase A, RNase U2, etc. : via 2’-3’ cyclic phosphate intermediate
- restriction enzymes (see Part prof. Muyldermans)
coupling enzymes
- DNA ligases:T4 DNA ligase (cofactor ATP), E. coli DNA ligase (cofactor NAD+)
substrate = ds, 5’-phosphate, 3’-OH, sticky ends versus blunt ends
- RNA ligase :
T4 RNA ligase (cofactor ATP)
substrate = ss (donor + acceptor), 5’-phosphate, 3’-OH
- topoisomerase I : from vaccinia virus
- TdT : terminal transferase : see above
G. Volckaert
Enzymatical modification of DNA, including PCR
16/02/2016
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Synthesis and amplification of DNA
in vivo: see Chapter : DNA replication
in vitro enzymatic synthesis of DNA & polymerase chain reaction:
polymerases:
- general characteristics: stability, processivity, substrate sensitivity, accuracy
- template, substrate (nucleotide precursors)
- activities: polymerisation, exonucleolytic processes, strand displacement, …
- (DNA, RNA) polymerases (including reverse transcriptase)
- DNA polymerase I, Klenow fragment polymerase, T4 DNA polymerase,
T7 DNA polymerase, reverse transcriptase (+ RNaseH)
- thermoresistant polymerases
- Taq polymerase, Pfu polymerase, Vent polymerase, etc.
- applications :
- “nick translation” technique
- labelling and tagging techniques (see also Chapter: Chemical DNA synthesis)
PCR :
- 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
G. Volckaert
Enzymatical modification of DNA, including PCR
16/02/2016
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- 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
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, …
- 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, SOE (see Chapter: Site-specific mutagenesis)
- (semi-)quantitative PCR, competitive PCR, real-time PCR
- incorporation of nucleotide analogs and tagging (see above)
G. Volckaert
Enzymatical modification of DNA, including PCR
16/02/2016