Elsevier

Genomics

Volume 6, Issue 4, April 1990, Pages 593-608
Genomics

Automated DNA sequencing of the human HPRT locus

https://doi.org/10.1016/0888-7543(90)90493-EGet rights and content

Abstract

The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contigous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5′ and 15,238 bp 3′ to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3′ → 5′ = 77%, P < 0.005) and those sequences in the latter half of the locus oriented randomly (5′ → 3′ = 67%, P < 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.

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      Recent methods involving combined amplification further improved longer reads (Murphy et al., 2005; SenGupta and Cookson, 2010). The first automated fluorescent DNA sequencer utilized a paired-end sequencing approach to sequence the entire hypoxanthineguanine phosphoribosyltransferase (HPRT) gene (Edwards et al., 1990; Pareek et al., 2011). A slab gel electrophoresis system was utilized by ABI Prism 310, the first commercial DNA sequencer, in 1996.

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    Sequence data from this article have been deposited with the EMBL/GenBank Data Librarieus under Accession No. M26434. Programs described herein may be obtained from Peter Rice.

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