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I was having a terrible time trying to PCR amplify a region of DNA that is 62% G+C rich. Your new product, CleanAmp 7-deaza-dGTP Mix, made it a breeze and solved my problem. I have since recommended this to our collaborators and it is now an essential component in our protocol.

Jane Michalski
University of Maryland, School of Medicine

 

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

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Recently, TriLink developed CleanAmp® 7-deaza-dGTP, an elegant fusion of the secondary structure reducing nucleotide analog 7-deaza-dGTP and TriLink's CleanAmdNTP Hot Start technology.

Figure 6

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.

 

 

 

 

 

 

 

As depicted in the figure above, 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 Reads

GC-rich DNA targets can be difficult to sequence due to the high degree of intrastrand secondary structure formation. The quality of sequencing results for GC-rich regions can be compromised by loss of signal, band compressions and weak signal base calls. PCR amplification of these problematic targets with the CleanAmp 7-deaza-dGTP Mix prior to Sanger dideoxy sequencing can significantly improve the read quality along the entire sequence. The figure below displays the sequencing results of a 78% GC-rich target, PCR amplified with either standard 7-deaza-dGTP mix or the CleanAmp 7-deaza-dGTP Mix prior to sequencing.

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CleanAmp 7-deaza-dGTP Robust Sequencing Reads

The use of CleanAmp 7-deaza-dGTP Mix in a pre-sequencing PCR amplification of B4GN4 (Beta-1,4-N-acetyl galactosaminyltransferase) decreases background noise and improves the quality of base calling over the entire length of the target in downstream sequencing reactions.  An overview of the sequencing electropherogram and a zoom-in of regions 15-35 bp and 235-255 bp, respectively, are shown. The 78% GC-rich target was PCR amplified from 5 ng of Human genomic DNA using either the CleanAmp GC-rich 2X PCR Master Mix or an analogous mix prepared with a standard 7-deaza-dGTP mix. The resulting amplicons were submitted for Sanger dideoxy sequencing.


As shown in the figure above, a closer examination of a 20 base pair region with 85% GC content reveals that CleanAmp 7-deaza-dGTP Mix provides accurate base calls much sooner in the sequence lending to longer sequencing reads. In addition, the zoomed-in region demonstrates the ability of the CleanAmp 7-deaza-dGTP Mix to decrease background and improve Phred quality scores over a stretch with 90.5% GC content. Overall, the use of CleanAmp 7-deaza dGTP Mix affords more reliable quality sequence information throughout the entire GC-rich target. This technology is also available in a convenient master mix format; see the CleanAmp GC-rich 2X PCR Master Mix.

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