2 Enzymatic Modification of DNA and RNA
43
• lOx buffer (if necessary) is 0.33 M Tris acetate pH 8.0, 0.66 M potassium
acetate, 0.1 M magnesium acetate, 5 mM DTT, 1 mg/ml bovine serum
albumin. This buffer should be stored insmall aliquots at -20°C.
• dNTP solution: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP in
water. This solution should be stored insmall aliquots at -20°C.
• T4 polymerase (various suppliers; usually 1 U/J.ll)
• TE buffer
• Chaotropic buffer, silica-gel spin columns
Procedure
1. After completion of restriction enzyme digest, combine the following:
Protruding
DNA (restriction digest)
20 Jll
0.1-5 jlg
dNTP
2111
2 nmoles each dNTP
T4 DNA polymerase
2 111
2U
2. Incubate at l2°C for 30 min.
3. Inactivate the enzyme by heating to 75°C for 10 min.
4. Add TE buffer to 100 J.ll total volume.
5. Purify the DNA by using a chaotropic buffer/silica-gel spin column system according to the manufacturer's instructions.
Taq DNA polymerase
In the late 1970s, the first thermostable DNA polymerasewas isolated from
Thermus aquaticus, a bacterium dwelling in hot springs. The enzymes lacks
3' ----+5' proofreading activity. This results in a rather high error rate of w- 4 •
Nevertheless, Taq DNA polymerase found widespread use in the polymerase chain reaction due to its. Another use is in direct sequencing of DNA.
The enzyme is a 65 kD DNA-dependent DNA polymerase that works best at
70°C to 80°C. It requires Mg+ in mM concentrations. Several thermostable
polymerases from other microorganisms, as well as recombinant enzymes
with modifications for increased stability and decreased error rates are now
commercially available for a number of specific PCR applications.
3'termini