Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR
Section snippets
Cells and DNA
Human lymphoblast cell lines heterozygous (GM07425) and homozygous (GM07405) for the β-globin IVS 110 mutation were obtained from the Coriell Cell Repositories (Camden, NJ, USA) and prepared as described previously [33]. Single cells were transferred directly into PCR tubes containing 10 μl of a PCR-compatible, proteinase K-based lysis solution [34] and were incubated at 50 °C for 30 min and then at 95 °C for 10 min. Previously extracted DNA from frozen gastric biopsies [35] was obtained from a
Logic of LATE–PCR assay design for pyrosequencing
Although several previous reports [24], [25], [26] have described the use of dsDNA templates for pyrosequencing, most pyrosequencing is carried out using ssDNA templates to boost reaction efficiency by preventing complementary strands from competing with the sequencing primer for hybridization to the template strand. LATE–PCR generates more single-stranded product than double-stranded product, thereby making it an attractive method for generating pyrosequencing substrate without an additional
Discussion
As more is learned about the role of genetic variants in complex diseases and therapeutic responses in the emerging age of genomics-based medicine, robust and efficient platforms for routine genetic screening will be increasingly needed. Pyrosequencing has emerged over the past decade as a powerful new tool for quantitative genotyping, although this has remained largely a research platform. For realistic use in a clinical or field setting, genetic assays will need to be not only reliable but
Acknowledgments
We kindly recognize help provided by Karen Eldin, Ruth Ann Luna, and James Versalovic at Texas Children’s Hospital (Houston, TX, USA) as well as Rene Myers and Liying Yan at Biotage (Westborough, MA, USA). Funding for this project was provided by Brandeis University, with additional support being provided by NIH PO1 CA91955 and NIH P50 HG02360.
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