"Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments

• Published in: Journal of biomolecular techniques Vol. 28; no. 3; pp. 97 - 110
• Main Authors: Ebili, Henry O, Hassall, James C, Fadhil, Wakkas, Ham-Karim, Hersh, Asiri, Abutaleb, Raposo, Teresa P, Agboola, Ayodeji Johnson, Ilyas, Mohammad
• Format: Journal Article
• Language: English
• Published: United States Association of Biomolecular Resource Facilities 01.09.2017
• Subjects: DNA - genetics; DNA Primers - genetics; Exons - genetics; Genomics; High-Throughput Nucleotide Sequencing - methods; Humans; Mutation - genetics; Polymerase Chain Reaction - methods; squirrel primers; direct Sanger sequencing
• ISSN: 1524-0215; 1943-4731
• DOI: 10.7171/jbt.17-2803-001

Currently, short DNA segments of sub-100 bp can be sequenced either directly by next-generation sequencing and pyrosequencing, which are expensive, or indirectly, Sanger sequencing combined with the cumbersome and failure-prone plasmid cloning. To circumvent these issues, we have generated a novel sequencing-purposed PCR assay using long-tailed primers (squirrel primers) to Sanger sequence directly sub-100 bp genomic amplicons. Squirrel primers, 40-65 nt in length, were used to amplify 51-93 bp long genomic sequences of exons 2 and 3, exon 15, exon 20, and exon 3 from colorectal cancer (CRC) cell lines and preamplified clinical CRC samples with known mutation status by PCR. Following this, a short second pair of primers that bind at the 5' region of the long tails was used for sequencing on the 3130 × l ABI Prism Genetic Analyzer. The sequencing data were analyzed FinchTV software. High-quality sequencing data were obtained from 51 to 93 bp long genomic sequences with our novel PCR assay, with capture of all of the target sequences in all of the samples in both the forward and reverse directions and confirmation of the mutation status of the CRC samples. Whereas the sequencing quality was independent of the template type, it showed a squirrel primer tail length-dependent pattern. Our novel PCR assay for direct and targeted Sanger sequencing of short genomic segments has potential applications in focused molecular/genetic profiling of cancer in research and diagnostics fields in which fragmented DNA, such as circulating tumor DNA and archival tissue DNA, are used as starting templates.