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Enhancement of HLA class II PCR-SSP typing by addition of cresol red and sucrose to the amplification mixtures
158
Abstracts
C-7.4 #199
ENHANCEMENT OF HLA CLASS II PCR-SSP TYPING BY ADDITION OF CRESOL RED AND SUCROSE TO THE AMPLIFICATION MIXTURES. DR Wagenknecht, KM Green, and JA Mclntyre, Histocompatibility Laboratory, Methodist Hospital of Indiana, Indianapolis, IN. Low and high resolution HLA class II typings can be performed by using PCR with sequence specific primers (SSP) in 2 1/2 hours from the arrival of a specimen into the laboratory Allele-specific amplified DNA is detected subsequent to agarose gel electrophoresis. Low resolution DR & DQ typing kits from Dynal ® for PCR-SSP typing require 38 separate reaction mixtures. Additional mixes are required for high resolution typing. To decrease gel-loading time, we asked if the gel loading buffer could be added to the reaction mixtures before PCR. The components of conventional loading buffer (Ficoll, bromophenol blue, xylene cyanol) inhibit PCR. We therefore, substituted compounds that do not inhibit PCR (sucrose for Ficoll and the dyes with cresol red). Final concentrations in the reaction mixture were 6% sucrose and 0.1 mM cresol red. This modification decreased the gel loading time by 50%. An unexpected advantage of this modification was increased reproducibility in the quantity of PCR products from different primer mixes. PCR-SSP reactions involve multiplex PCR, i.e. positive control and allele-specific primer pairs in each mix. Certain HLA-DQ SSP mixes which previously had weak or no allele-specific amplification had satisfactory positive controls. By adding sucrose/cresol red, more allele specific PCR product was observed and specific SSP which had failed previously, now amplified. The mechanism for increase in PCR product is likely due to a sucrose induced increase in reaction mixture osmolarity. In summary, by adding sucrose/cresol the quality of PCR-SSP class II typings improved and turn-around time decreased.
C-7.4 #200
PERFORMANCE OF A PROTOTYPE HLA-DRB GENERAL TYPING KIT IN 9 NORTH AMERICAN LABS. 2tB._.B.e, gg..v.i.~, CA Aldrich, SF Boyle, JF Novotny Jr., Departments of Human Genetics & PCR Diagnostic Development, Roche Molecular Systems, Inc., Alameda, CA & Sommerville, NJ. To simplify PCR/oligonucleotide probe typing for the HLA-DRB loci, a reverse dotblot kit has been developed. This kit provides standardized reagents for every step of the procedure - from DNA extraction through PCR DNA amplification and detection - and takes approximately four and one half hours to run. In addition, dUTP and AmpEraseTM (Uracil-N-glycosylase) have been incorporated into the amplification reaction to prevent PCR-product carryover. The strip contains 12 sequence specific oligonucleotide probes (including one control probe) and is designed to distinguish 16 groups of DRB 1 alleles. In order to assess the performance and utility of this kit in the clinical lab, nine labs within North America were asked to participate in pre-clinical trials. Each lab compared the prototype kit to their current in-house DNA-based technology which included PCRSSOP (7 labs), PCR-SSP (1 lab), and PCR-RFLP (1 lab). Two labs also provided serologic data. As of this date, data has been received from eight of the nine labs; these results show over 99.9% concordance. All 717 samples typed by PCR-SSOP and 212 samples typed by PCR-SSP were concordant with the prototype reverse dot-blot kit. Ninety-five out of 96 samples typed by the PCR-RFLP technique were concordant. The one discordant sample is being sequenced to determine the correct HLA-DRB 1 genotype. These data show the prototype kit to be highly reliable and easy to run in a clinical setting.
Abstracts
C-7.4 #199
ENHANCEMENT OF HLA CLASS II PCR-SSP TYPING BY ADDITION OF CRESOL RED AND SUCROSE TO THE AMPLIFICATION MIXTURES. DR Wagenknecht, KM Green, and JA Mclntyre, Histocompatibility Laboratory, Methodist Hospital of Indiana, Indianapolis, IN. Low and high resolution HLA class II typings can be performed by using PCR with sequence specific primers (SSP) in 2 1/2 hours from the arrival of a specimen into the laboratory Allele-specific amplified DNA is detected subsequent to agarose gel electrophoresis. Low resolution DR & DQ typing kits from Dynal ® for PCR-SSP typing require 38 separate reaction mixtures. Additional mixes are required for high resolution typing. To decrease gel-loading time, we asked if the gel loading buffer could be added to the reaction mixtures before PCR. The components of conventional loading buffer (Ficoll, bromophenol blue, xylene cyanol) inhibit PCR. We therefore, substituted compounds that do not inhibit PCR (sucrose for Ficoll and the dyes with cresol red). Final concentrations in the reaction mixture were 6% sucrose and 0.1 mM cresol red. This modification decreased the gel loading time by 50%. An unexpected advantage of this modification was increased reproducibility in the quantity of PCR products from different primer mixes. PCR-SSP reactions involve multiplex PCR, i.e. positive control and allele-specific primer pairs in each mix. Certain HLA-DQ SSP mixes which previously had weak or no allele-specific amplification had satisfactory positive controls. By adding sucrose/cresol red, more allele specific PCR product was observed and specific SSP which had failed previously, now amplified. The mechanism for increase in PCR product is likely due to a sucrose induced increase in reaction mixture osmolarity. In summary, by adding sucrose/cresol the quality of PCR-SSP class II typings improved and turn-around time decreased.
C-7.4 #200
PERFORMANCE OF A PROTOTYPE HLA-DRB GENERAL TYPING KIT IN 9 NORTH AMERICAN LABS. 2tB._.B.e, gg..v.i.~, CA Aldrich, SF Boyle, JF Novotny Jr., Departments of Human Genetics & PCR Diagnostic Development, Roche Molecular Systems, Inc., Alameda, CA & Sommerville, NJ. To simplify PCR/oligonucleotide probe typing for the HLA-DRB loci, a reverse dotblot kit has been developed. This kit provides standardized reagents for every step of the procedure - from DNA extraction through PCR DNA amplification and detection - and takes approximately four and one half hours to run. In addition, dUTP and AmpEraseTM (Uracil-N-glycosylase) have been incorporated into the amplification reaction to prevent PCR-product carryover. The strip contains 12 sequence specific oligonucleotide probes (including one control probe) and is designed to distinguish 16 groups of DRB 1 alleles. In order to assess the performance and utility of this kit in the clinical lab, nine labs within North America were asked to participate in pre-clinical trials. Each lab compared the prototype kit to their current in-house DNA-based technology which included PCRSSOP (7 labs), PCR-SSP (1 lab), and PCR-RFLP (1 lab). Two labs also provided serologic data. As of this date, data has been received from eight of the nine labs; these results show over 99.9% concordance. All 717 samples typed by PCR-SSOP and 212 samples typed by PCR-SSP were concordant with the prototype reverse dot-blot kit. Ninety-five out of 96 samples typed by the PCR-RFLP technique were concordant. The one discordant sample is being sequenced to determine the correct HLA-DRB 1 genotype. These data show the prototype kit to be highly reliable and easy to run in a clinical setting.