A novel, one-step amplification and oligonucleotide ligation procedure for multiplex genetic typing

Journal Article · Thu Sep 01 04:00:00 EDT 1994 · American Journal of Human Genetics

OSTI ID:134003

Eggerding, F A ^[1]

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1. Perkin Elmer Corporation, Foster City, CA (United States)

A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method which combines in one assay DNA amplification by the polymerase chain reaction (PCR) with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I target DNA is amplified using high-melting primers in a two-step PCR cycle that employs a 72{degrees}C anneal-elongation step. In stage II genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes located between PCR primers is accomplished by several cycles of denaturation at 94{degrees}C followed by anneal-ligation at 55{degrees}C. Ligation products are fluorochrome-labeled at their 3{prime}-ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used for multiplex detection of 30 cystic fibrosis mutations and for analysis of ras gene point mutations. Because mutation detection occurs concurrently with target amplification, the technique is rapid, highly sensitive and specific, easily automatable, and requires minimal sample processing.

• OSTI ID: 134003
• 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

Related Subjects

44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
55 BIOLOGY AND MEDICINE
BASIC STUDIES
AUTOMATION
DETECTION
DNA HYBRIDIZATION
DNA SEQUENCING
ELECTROPHORESIS
FLUORESCENCE
GENE MUTATIONS
GENES
GENOTYPE
MULTIPLEXERS
OLIGONUCLEOTIDES
POLYMERASE CHAIN REACTION
PROBES
SAMPLE PREPARATION
SENSITIVITY
SPECIFICITY
STRUCTURAL CHEMICAL ANALYSIS