Automated DNA sequencing methods involving polymerase chain reaction
| Autor: | M N Kronick, W Salser, Michael W. Hunkapiller, P E Mayrand, Lincoln J. Mcbride, S.M. Koepf, Richard A. Gibbs |
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| Rok vydání: | 1989 |
| Předmět: | |
| Zdroj: | Clinical Chemistry. 35:2196-2201 |
| ISSN: | 1530-8561 0009-9147 |
| Popis: | Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype. |
| Databáze: | OpenAIRE |
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