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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Ligase chain reaction using colorimetric detection
Layne Huiet, Annette Tumolo, Luis Ugozzoli, Tony Reyes, Jimmie Lowry, Bruce Wallace and Frank Witney,
Bio-Rad Laboratories, Hercules, CA 94547
Ligase chain reaction is an amplification method which involves the ligation of two sets of adjacent oligonucleotides. These pairs of oligonucleotides are complementary to one another and therefore provide the templates for the exponential amplification of the products after repeated rounds of ligation. Use of a thermostable DNA ligase allows the reaction to be cycled in conventional thermocyclers. The specificity of the reaction is such that only oligonucleotides which hybridize without mismatch at their junction are ligated. This requirement for exact base pairing can be used to discriminate single base differences in a target sequence. Here we will present a method for carrying out LCR reactions and the nonradioactive detection of the products of the reaction using both a synthetic model and a human genome model. This is carried out by binding the LCR reaction products containing a biotin moiety to a streptavidin coated microtiter well. An alkaline phosphatase/oligonucleotide conjugate is hybridized to the ligation products bound to the wells and utilized as the reporter molecule with NADPH or pNPP as the substrate. This system has the advantage of a nonradioactive detection which is not gel based, lending itself to automation.
This work is not supported by DOE