GC-RICH PCR System Troubleshooting

Troubleshooting GC-Rich Templates

Template quality significantly influences PCR outcome.

For the amplification of GC-rich targets up to 5 kb in length, DNA with repetitive sequences, and DNA mixtures with varying GC content, Roche recommends using the GC-RICH PCR System or the GC-RICH PCR System, dNTPack. The GC-RICH PCR System includes three specially formulated components not included in most PCR systems:

1. Special enzyme mix
2. Reaction buffer, both containing detergents and DMSO
3. Special GC-RICH Resolution Solution

The optimal reaction conditions (incubation times and temperatures, concentration of GC-RICH PCR System enzyme mix, GC-RICH resolution solution, template DNA, Mg²⁺) depend on the template/primer pair and must be determined for each assay. For difficult templates the titration of GC-RICH Resolution Solution may be required (0.5 to 2.5 M, in steps of 0.25 M).

In addition to the above, also consider the following options to optimize PCR using difficult templates:

• Add DMSO (v/v) from 2 to 10%; Note that at concentrations higher than 5% DMSO, DNA polymerase activity may be reduced. A concentration of 10% DMSO inhibits the DNA polymerase. Please also note: Adding DMSO can also negatively influence the error rate of the PCR.
• Additional options include:
  • Add glycerol (5 to 25%)
  • Add betain (0.5 to 2 M)
  • Add urea
• Linearize using a restriction enzyme when the DNA template is a plasmid.