Sequencing telomeric DNA template with short tandem repeats using dye terminator cycle sequencing.
The standard DNA sequencing methods for use in conjunction with commercially available sequencing kits are effective in sequencing a majority of templates. However, templates rich in dinucleotide and tetranucleotide repeats and a telomeric DNA containing tandem repeats are difficult to sequence adequately using these methods. Base compression artifacts due to formation of secondary structure on the nascent strands and slippage of the DNA polymerase accompanied by premature chain termination in homopolymer and short tandem repeat regions of DNA are commonly encountered problems in sequencing core laboratories. In an attempt to sequence such repeat regions of telomeric DNA templates using dye terminator chemistry, we investigated the effect of increasing the annealing time and temperature in combination with the use of denaturing conditions. Specifically, we compared the commonly used ABI PRISM BigDye, dGTP BigDye, and DYEnamic ET terminator chemistries for sequencing telomeric DNA templates rich in CA- and AACCCC-type repeats and for sequencing a template rich in dinucleotide (GT and CT) and tetranucleotide repeats. The routine reaction protocol was modified by adding either 1 M 1-carboxy-N,N,N-trimethylmethanaminium inner salt (betaine) or 5% dimethyl sulfoxide (DMSO) as denaturants in the reaction mixture. In addition, the annealing and denaturation times were increased to allow successful primer extension for linear growth of sequencing reaction product. Many of the artifacts in sequencing are known to be due to reduced stability of the hybrid formed between the template and the nascent strand. The effects of using denaturants to break secondary structures in the nascent chain and of increasing the denaturation and annealing times are discussed.We were able to sequence DNA templates with tandem repeats that failed to sequence under routine reaction conditions.
Zhao
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Molecular Biotechnology Core, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Haqqi
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Yadav
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