Improved Cycle Sequencing of GC-Rich Templates by a Combination of Nucleotide Analogs

Abstract

A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution

“…Deoxyinosine is a well-studied universal base with a strong preference for base pairing with cytosine (11,16). It is used as a replacement for guanine in DNA sequencing (17,18). Terminal transferase is also known to incorporate universal bases with unbiased mutational spectra such as 3-nitropyrrole and 5-nitroindole (19,20).…”

Hot Start 7-Deaza-dGTP Improves Sanger Dideoxy Sequencing Data of GC-Rich Targets

“…The use of this nucleotide analog effectively linearizes the DNA sequences in preparation for Sanger dideoxy sequencing by destabilizing secondary structures such as G-quadruplexes. This allows the DNA fragments to migrate more predictably through the polyacrylamide gel and reduces the possibility of ambiguous base calls in sequencing results (Motz, Svante et al, 2000). CleanAmp™ dNTPs also offer the added benefit of reduced off-target amplification due to Hot Start activation in the PCR assay.…”

“…Additives such as dimethyl sulfoxide (DMSO), betaine, formamide, and glycerol are often added into sequencing reactions to reduce secondary structure and to promote strand separation (Jung et al, 2002). It is well known that the use of modified analogs such as 7-deaza-dGTP (7-deaza-2-deoxyguanosine-5-triphosphate) and dITP (2-deoxyinosine-5-triphosphate) can be used in the sequencing reaction in place of dGTP or in some combination thereof to reduce secondary structure (Motz, Svante et al, 2000). 7-deaza-dGTP is a modified dGTP analog which lacks a nitrogen at the seven position of the purine ring.…”

7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands

“…Therefore, many manufacturers add deaza analogues to their sequencing reaction mixes and in situations where commercial kits are not available the addition of deaza-dGTP and deaza-dITP is of great benefit. 9 If DNA is isolated from formaldehyde fixed paraffin wax embedded tissue sections using microdissection poor quality and low amounts of DNA become an additional issue. The use of deaza-dGTP has been reported to improve PCR product yield, 10 and this is confirmed by our study for the generation of PCR products from the p16 INK4A promoter.…”