Polymerase chain reaction (PCR) is a widely applied approach in molecular biology. DNA templates with high GC content and short repeats sometimes cause troubles because the formation of secondary structures that resist denaturation makes GC-rich sequences difficult to amplify by PCR.

For this purpose Kapa Biosystems is recommending the KAPA2G Robust HotStart PCR kit designed for robust amplification of difficult templates. The kit consists of KAPA2G Robust Hotstart DNA Polymerase, three KAPA2G buffers optimized for specific applications, and KAPAEnhancer 1, a proprietary PCR additive which improves amplification of some, but not all, primer/template combinations. Three reaction conditions are recommended for GC-rich PCR specifically, namely KAPA2G GC Buffer, KAPA2G GC Buffer supplemented with 4% DMSO, and KAPA2G Buffer A, supplemented with 1x KAPAEnhancer 1 and 5% DMSO. Recommended extension times vary from 15 to 60 seconds per kb, depending on the template/primer combination used.

Here we will show the influence of these different buffers to GC rich DNA: 

1.    Reaction setup

Three sets of KAPA2G Robust HotStart reactions were performed, according to the recommendations for GC-rich PCR in the KAPA2G Robust HotStart Technical Data Sheet. These recommendations are: KAPA2G GC Buffer; KAPA2G GC Buffer and 4% DMSO; and KAPA2G Buffer A, KAPAEnhancer 1 and 5% DMSO. Reactions were supplemented with MgCl₂ to a final concentration of 2 mM. Human genomic DNA (10 ng per 25 μL reaction) was used as template. See tables below for detailed reaction setup. Half of each reaction product was analyzed in a 1% agarose-TBE gel and visualised by ethidium bromide staining. GeneRuler (Fermentas; 5 μL per lane) was used as marker.

Kapa 2G  GC Buffer:

Kapa 2G  GC Buffer + 4% DMSO:

Kapa 2G Buffer A + Enhancer1 + 5% DMSO:

2. Conclusions from these experiments are the following:

KAPA2G Robust HotStart successfully amplified all ten of the GC-rich amplicons with small amounts of non-specific products. High yields were achieved for all ten amplicons with at least one of the reaction setup conditions tested. In general, the best results were achieved with KAPA2G Buffer A, supplemented with 1x KAPAEnhancer 1 and 5% DMSO.

In comparison to other enzymes in the market, we can state that many suppliers attempt to improve robustness and speed of wild-type polymerases by supplying excessive amounts of enzyme, but it is clear that these overformulated enzymes still do not exhibit the superior performance of KAPA2G Robust Hostart (Data not shown here but available on request).

Due to the high processivity of the KAPA2G Robust HotStart DNA polymerase, good amplification was observed with an extension time of only 30 seconds per kb. For some of these amplicons, extension times may be reduced to 15 seconds without compromising yield and specificity.

Amplicon details: