Research Update
Amplifying GC-rich Sequences
DNA targets high in GC content have a tendency to form a high degree of intramolecular secondary structure such as hairpins and G quadruplexes. To amplify GC-rich sequences by PCR and reduce the probability of forming off-target products, these secondary structures must be broken up to allow for adequate template separation. 7-deaza-dGTP is a secondary structure reducing dGTP analog that lacks a nitrogen at the 7 position of the purine ring. Loss of this nitrogen disrupts Hoogsteen bonding which prevents G quadruplexes from forming, while not interfering with standard Watson-Crick base pairing. Incorporation of 7-deaza-dGTP into a PCR reaction reduces secondary structure formation and allows for adequate amplification of GC-rich regions of DNA. Detection of GC-rich targets is becoming increasingly important for the molecular diagnosis of inherited and deadly diseases.
TriLink’s high quality 7-deaza-dGTP is now available at an even lower price. In addition to 7-deaza-dGTP, CleanAmp™ 7-deaza-dGTP, TriLink’s Hot Start version of 7-deaza-dGTP is available for better PCR amplification of especially problematic situations such as very high GC-content DNA, very limited samples or extensively degraded DNA.
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Technology Spotlight
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 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.
NTPs for Sequencing
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Ask An Expert Featured Question
Q -
Which of your modified nucleoside triphosphates can be incorporated into DNA molecules through PCR?
A –
Aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 7-deaza-dGTP, and 7-deaza-dATP, 5-Methyl-dCTP, 5-Iodo-dUTP, 5-Bromo-dUTP, 5-Fluoro-dUTP, N4-methyl-dCTP, 5-propynyl-dUTP and 5-propynyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and alpha-thio-dNTPs can be incorporated through PCR. As not all of these analogs will incorporate with similar efficiencies in PCR, some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.
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