Robust Amplification of GC-Rich Targets
Struggling with amplification of DNA targets high in GC content? PCR product formation can often be compromised by inadequate strand separation and the propensity for complex secondary structure formation. The use of standard 7-deaza-dGTP is a notable method for overcoming this problem.1 Recently, TriLink developed CleanAmp™ 7-deaza-dGTP, an elegant fusion of the secondary structure reducing nucleotide analog 7-deaza-dGTP and TriLink's CleanAmp™ dNTP Hot Start technology.
As depicted in the figure to the right, when targets with increasing GC content are amplified, the use of the CleanAmp™ 7-deaza-dGTP Mix consistently provides a clean, high-yield product. For the 79% GC-rich GNAQ target, it is not possible to form the desired amplicon without the use of the CleanAmp™ 7-deaza-dGTP Mix. CleanAmp™ 7-deaza-dGTP is available individually for amplification of routine GC-rich targets or as the CleanAmp™ 7-deaza-dGTP Mix which is recommended for more challenging targets with higher GC content.
Improving Downstream Sequencing
GC-rich DNA targets can be difficult to sequence due to the high degree of intrastrand secondary structure formation. Sequencing GC-rich regions can also be complicated by loss of signal, band compressions and weak signal base calls. Pre-amplification of these problematic targets using CleanAmp™ 7-deaza-dGTP prior to sequencing can significantly improve the read quality. Complete substitution of CleanAmp™ 7-deaza-dGTP for dGTP in the pre-PCR step affords the best sequencing results.
As shown in the figure to the right, even with the addition of standard 7-deaza-dGTP into the pre-amplification reaction, this troublesome 641 bp target cannot be sequenced past 310 bp. In comparison, the introduction of CleanAmp™ 7-deaza-dGTP nearly doubles the number of correct base calls.
The detailed sequence read shows a 60 bp stretch in which samples pre-amplified with standard 7-deaza-dGTP performed the strongest. In this 80% GC-rich stretch, reactions employing CleanAmp™ 7-deaza-dGTP substantially improved base call quality compared to reactions with standard 7-deaza-dGTP. |
1 McConlogue, L., Brow, M.A. and Innis, M.A. (1988) Structure independent DNA amplification by PCR using 7-deaza-2’-deoxyguanosine. Nucleic Acids Res, 16, 9869.
 Click image to enlarge
The use of CleanAmp™ 7-deaza-dGTP in a pre-sequencing PCR step improves the quality of base calling and length of reads in downstream sequencing reactions. |
