The
creation of unnatural base pairs (UBPs) has rapidly advanced
the genetic alphabet expansion technology of DNA, requiring a new
sequencing method for UB-containing DNAs with five or more letters.
The hydrophobic UBP, Ds–Px, exhibits high fidelity in PCR and
has been applied to DNA aptamer generation involving Ds as a fifth
base. Here, we present a sequencing method for Ds-containing DNAs,
in which Ds bases are replaced with natural bases by PCR using intermediate
UB substrates (replacement PCR) for conventional deep sequencing.
The composition rates of the natural bases converted from Ds significantly
varied, depending on the sequence contexts around Ds and two different
intermediate substrates. Therefore, we made an encyclopedia of the
natural-base composition rates for all sequence contexts in each replacement
PCR using different intermediate substrates. The Ds positions in DNAs
can be determined by comparing the natural-base composition rates
in both the actual and encyclopedia data, at each position of the
DNAs obtained by deep sequencing after replacement PCR. We demonstrated
the sequence determination of DNA aptamers in the enriched Ds-containing
DNA libraries isolated by aptamer generation procedures targeting
proteins. This study also provides valuable information about the
fidelity of the Ds–Px pair in replication.
The autosomal dominantly inherited spinocerebellar ataxias (SCAs) can be caused by dynamic mutations of short tandem repeats within various genes. Because of the significant clinical overlap among the various SCA types, molecular screening of multiple genetic loci by fluorescent PCR and capillary electrophoresis is necessary to identify the causative repeat expansion. We describe a simple, rapid, and inexpensive strategy to screen for CAG repeat expansion mutations at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Plasmid DNAs of known repeat sizes were used to generate threshold melt peak temperatures, which rapidly and effectively distinguish samples carrying an expanded allele from those carrying nonexpanded alleles. Melting curve analysisepositive samples were confirmed by capillary electrophoresis sizing of the triplet-primed PCR products. All three assays achieved 100% sensitivity, with 95% CIs of 67.86% to 100% (SCA1), 74.65% to 100% (SCA2), and 91.58% to 100% (SCA3). The SCA1 assay also achieved 100% specificity (95% CI, 97.52%e100%), whereas the SCA2 and SCA3 assays achieved specificity of 99.46% (95% CI, 96.56%e99.97%) and 99.32% (95% CI, 95.70%e99.96%), respectively. These screening assays provide robust and highly accurate detection of expanded alleles and are amenable to large-scale screening while minimizing the need for capillary electrophoresis sizing for every sample.
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