A rapid technique to develop CA repeats and STSs from BACs by direct cycle-sequencing
Journal Article
·
· American Journal of Human Genetics
OSTI ID:134061
- UCLA School of Medicine, Los Angeles, CA (United States)
CA microsatellites and sequence-tag-sites (STSs) are crucial to mapping strategies employed in many laboratories. Described here is a method to quickly generate CA repeats and STSs by cycle-sequencing from bacterial artificial chromosomes (BACs). Commonly, cosmids have been used as templates for cycle-sequencing, but an individual cosmid may not be large enough to contain a CA repeat. However, BACs contain human genomic DNA fragments of several hundred kilobases (up to 300 kilobases); thus, given the frequency of CA repeats in the human genome, each BAC may contain one or more CA repeat sequence(s). Primers, designed to detect the ends of CA repeats ((CA)7(A/G/T)N), with BACs as template, are used in cycle-sequencing reactions to sequence the flanking regions. Once the CA repeat flanking sequence is obtained, then specific primers are made so that: (1) the CA repeat itself may be sequenced through to determine the sequence on the other flanking side of the CA and a reverse primer designed so that the CA repeat may be PCR amplified and assayed for polymorphism; or (2) any unique region flanking the CA repeat may also be PCR amplified and used as an STS.
- OSTI ID:
- 134061
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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