25/03/11 - 7-Deaza-dGTP for Sequencing Difficult Templates

PCR-based sequencing can encounter regions of DNA that are problematic due to either inadequate PCR amplification or sequence motifs in PCR-amplified DNA that prevent sequencing. Certain sequences can also foil sequencing of regions within clonally amplified DNA.

From a comprehensive technical review (1) of methods for PCR amplification of these “difficult templates,” which include GC-rich regions, hairpins and long stretches of G, one notable solution recommends using 7-deaza-dGTP in place of dGTP. When 7-deaza-dGTP is used in PCR, amplicon yield is improved and the resultant 7-deaza-dG-containing amplicons sequence is better.

Research by Jung et al (2) illustrates how 7-deaza-dGTP enables both PCR amplification and sequencing of GC-rich CpG islands, which are currently of widespread interest in epigenetics and cancer research. These investigators also suggested that addition of 7-deaza-dGTP is particularly useful when small amounts of poor quality DNA are available as starting material, which is frequently the case for formalin-fixed paraffin-embedded (FFPE) cancer biopsy specimens.

This method has also been used in whole-genome sequencing during PCR-mediated gap-closure of cosmid clones (3). In addition, dITP can be used with 7-deaza-dGTP to sequence difficult templates according to the method described by Motz et al. (4) wherein a 4:1 mixture of 7-deaza-dGTP : dITP is used for PCR to give doubly-modified amplicons for sequencing.

We also offer Hot start CleanAmp 7-deaza-dGTP for PCR.