The ability to use crude samples as templates in PCR eliminates the need for laborious and costly DNA extraction procedures and allows for faster turnaround times from sampling to results. The feasibility of high-throughput or routine crude sample PCR – particularly for diagnostic applications – has remained low due to the fact that wild-type Taq is easily inhibited by cellular debris, yielding low success rates and inconsistent results.
KAPA2G Robust DNA Polymerase is a novel DNA polymerase derived from wild-type Taq through molecular evolution, and is specifically designed for high performance PCR in chemically complex environments. The enzyme is resistant to many inhibitors commonly found in crude samples (including denatured protein, salts and some metabolites) and is ideally suited for the routine amplification of DNA fragments ≤1 kb from crude samples such as:
- saliva or buccal swabs
- tissue samples, e.g. mouse ear or tail clippings
- bacterial and yeast colonies/cultures
- cultured mammalian cells
- Arabidopsis leaf discs
Crude sample PCR with KAPA2G Robust typically requires an initial denaturation of 5 – 10 min at 95 °C to release DNA for amplification. For some sample types, results may be improved by performing this step in a suitable buffer, spinning down cellular debris and using cleared supernatants in the PCR. Since extension times of 15 – 30 sec/kb per cycle are sufficient for most template types, turnaround times from sample to results can be reduced significantly. KAPA2G Robust and KAPA2G Robust HotStart are supplied with three buffers and a proprietary enhancer, which offer extensive optimization options for different crude sample types. In addition, KAPA2G Robust is compatible with most common PCR additives and with routinely used cell lysis agents and protocols.