DNA sequencing strategy that requires only five bases of known terminal sequence for priming

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 22; pp. 10162 - 10166
• Main Authors: Fu, D.J, Broude, N.E, Koster, H, Smith, C.L, Cantor, C.R
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 24.10.1995; National Acad Sciences; National Academy of Sciences
• Subjects: Annealing; Base Sequence; Biochemistry; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA primers; DNA Primers - chemistry; DNA probes; DNA, Single-Stranded - chemistry; Enzymes; Genetic Techniques; Indicators and Reagents; Magnetics; man; Molecular Sequence Data; Oligonucleotides; Polymerase chain reaction; Polymerase Chain Reaction - methods; positional sequencing by hybridization; sequence analysis; Sequencing; sequencing primers; Single stranded DNA; single stranded dna targets; Walking
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.22.10162

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.