A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing.

Abstract

A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed that allows for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method that combines in one assay DNA amplification by 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 (Tm values between 68 degrees C and 89 degrees C) in a two-step PCR cycle that employs a 94 degrees C denaturation step and a 72 degrees C anneal-elongation step. In stage II, genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes (Tm values between 51 degrees C and 67 degrees C) located between the 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' ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used successfully to analyze human genomic DNA for cystic fibrosis (CF) alleles. Because product detection occurs concurrently with target amplification, the technique is rapid, highly sensitive, and specific and requires minimal sample processing.