Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR
Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-e...
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Veröffentlicht in: | Analytical biochemistry 2006-06, Vol.353 (1), p.124-132 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)–PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for β-thalassemia. |
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ISSN: | 0003-2697 1096-0309 |
DOI: | 10.1016/j.ab.2006.02.012 |