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Digestion of PCR Products

• Cleavage of PCR Products Directly after Amplification Reaction (see below)
• Cleavage Efficiency Close to the Termini of PCR Fragments

In experiments with amplified DNA, restriction endonuclease (RE) digestion is usually performed directly in the PCR mixture, without time-consuming and expensive purification steps.

The majority of Fermentas restriction enzymes show sufficient activity (>20%) in PCR buffers (traditional PCR buffer and PCR buffer with (NH₄)₂SO₄); some require the addition of their optimal buffer to obtain adequate activity. Direct digestion of amplified product is not recommended for only 9 of 162 Fermentas enzymes tested (see Recommendations below).

None of the PCR mixture components, including primers, dNTP's, template DNA and Taq DNA Polymerase affect RE activity. In some cases, a RE having 100% activity in the initial PCR mixture is not able to digest the PCR product, even after addition of extra amounts of the RE or its optimal buffer. There have been reports in the literature on the inability of some REs to digest DNA following PCR, however, these data are contradictory (1, 2). According to our data, the same enzymes from different companies behave in the same way but their ability to digest the PCR product differs, depending on the nature of template DNA and primers used. In these cases, thorough purification of the PCR product may help. We have found that after three-fold dilution of the PCR mixture with RE reaction buffer, the DNA was digested by most REs, though in some cases a large excess of enzyme or prolonged incubation time was necessary.

Recommendations

• Even if the RE works well in the PCR buffer, we still recommend three-fold dilution of the PCR mixture after amplification and prior to digestion, in order to avoid digestion problems. The optimal RE reaction buffer is suitable for dilution, but usually it is more convenient to use either 1X or 2X Y+/Tango™ buffer, choosing the concentration that ensures higher activity of enzyme and lower potential star activity.
• Due to their low activity in PCR buffer, direct digestion of amplified DNA without dilution of reaction mixture or purification of the PCR fragment is not recommended for AarI, AatII, BseSI, CfrI, Cfr9I, Cfr42I, HphI, ScaI and SmiI. BglI, BfuI, BplI, BspTI, EcoRI, FspAI, Hin4I, Mva1269I, PauI and SalI require the addition of their 10X optimal buffers to a final 1X concentration in the PCR mixture, prior to digestion.
• For cloning applications, we do not recommend direct digestion of the amplified fragment in the PCR mixture without purification, since Taq DNA Polymerase is still active and may result in blunting of 5'-sticky ends and the addition of an extra dA nucleotide to the blunt ends of restriction fragments.

References

1. Blanck, A., et al., Activity of restriction enzymes in a PCR mix, Biochemica, Boehringer Mannheim GmbH, 2, 14, 1995.
2. Eastlake, P., et al., in PCR Essential Data (Newton, C.R. ed.), John Willey & Sons Inc., Chichester, New York, Brisbane, Toronto, Singapore, 143-148, 1995.

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Updated September 20, 2002 10:46