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DNA typing of cadaveric donors by a rapid sequence specific oligonucleotide probe typing kit
Abstracts
157
C-7.4 #197
DNA TYPING OF CADAVERIC DONORS BY A RAPID SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBE TYPING KIT. R Cirocco, M Markou, K Zucker, C Gomez, V Esquenazi, and J Miller, Dept. Surgery, Div. Transplantation, Univ. Miami Med. Sch,Miami, FL, and the Miami Veterans Admin. Hosp, Miami, FL. Classical Sequence Specific Oligonucleotide Probe (SSOP) typing has the advantage of high resolution due to multiple probes and overlapping sequence analysis. Its disadvantages are it is time consuming (3 to 10 days), labor intensive (30 to 60 individual hybridizations), and can be difficult to interpret. Recently, a medium resolution SSOP kit has become available from Biotest (Denville, NJ). This procedure links the DRB amplicon in a microtiter plate to a membrane. Due to the fact that the amplicon is isolated in one well, after denaturation the 28 different probes can be added (1 to each well) in one facile procedure. The result is a medium resolution SSOP typing procedure with one hybridization/color development protocol. Briefly, the Biotest procedure is as follows: The DNA is isolated from whole blood by hypotonic lysis of RBC's and membranes digested by proteinase K. The proteinase K is inactivated and 5 ul of the DNA rich supernatant is PCR amplified in a 30 cycle amplification. With cadaver donors, an agarose gel electrophoresis is necessary to assure amplification. The amplicon (2 ul) is placed in each of the 28 wells, dried at 60°C for 10 minutes, and denatured. Hybridization is accomplished with the probe sets at 46°C, followed by several washes. The conjugate is added, followed by color development. The resulting black dots are scored and allelic patterns resolved. Twelve cadaver donors were DNA typed by the Reverse Dot Blot Procedure for DRB1,3,4,5, developed and proven accurate by our laboratory, and with the SSOP kit. Concordance was 100% between the two methods. There were eleven heterozygotes and one homozygote. Eleven of twelve gave group-specific resolution with both tests, while one individual gave high resolution with the SSOP. In summary, the SSOP kit is facile, rapid, and allows typing of cadaveric donors in a timely fashion.
C-7.4 #198
THE USE OF DRIED BLOOD STAINS AS A N A R C H I V A B L E SPECIMEN FOR CLASS II HLA TYPING BY THE POLYMERASE CHAIN REACTION. U Heine, E. Owens, J. Medley, K. VonCannon, and JM Mason, Department of HLA/Paternity, Roche Biomedical Laboratories, Burlington, NC. Dried blood stains ("DBS")were e v a l u a t e d as potential specimens for DNA typing by PCR. The stability of such specimens over time was evaluated in two independent studies. First, 27 month old DBS were a m p l i f i e d with primers to the p o l y m o r p h i s m at DISS0. These specimens had been subjected to this assay as part of a p a t e r n i t y test panel in 1991. These specimens were then typed for HLA DRB and DQB by PCR/SSOP. All 22 specimens in this study were suitable for typing at all three loci. Second, Fresh blood was collected from twenty u n r e l a t e d individuals into N a : H e p a r i n tubes and typed for DRB by standard serology. DBS were then prepared and immediately typed for DRB by PCR. The PCR assay was repeated p e r i o d i c a l l y over time as part of an ongoing 5 year study. Only two discrepancies were noted in the entire study, and these were between serological and DNA typing. DBS were found to be stable at room temperature for the entire period of this study and p r o v i d e d reproducible results with all anticoagulants evaluated (Na:Heparin, ACD, and EDTA)
157
C-7.4 #197
DNA TYPING OF CADAVERIC DONORS BY A RAPID SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBE TYPING KIT. R Cirocco, M Markou, K Zucker, C Gomez, V Esquenazi, and J Miller, Dept. Surgery, Div. Transplantation, Univ. Miami Med. Sch,Miami, FL, and the Miami Veterans Admin. Hosp, Miami, FL. Classical Sequence Specific Oligonucleotide Probe (SSOP) typing has the advantage of high resolution due to multiple probes and overlapping sequence analysis. Its disadvantages are it is time consuming (3 to 10 days), labor intensive (30 to 60 individual hybridizations), and can be difficult to interpret. Recently, a medium resolution SSOP kit has become available from Biotest (Denville, NJ). This procedure links the DRB amplicon in a microtiter plate to a membrane. Due to the fact that the amplicon is isolated in one well, after denaturation the 28 different probes can be added (1 to each well) in one facile procedure. The result is a medium resolution SSOP typing procedure with one hybridization/color development protocol. Briefly, the Biotest procedure is as follows: The DNA is isolated from whole blood by hypotonic lysis of RBC's and membranes digested by proteinase K. The proteinase K is inactivated and 5 ul of the DNA rich supernatant is PCR amplified in a 30 cycle amplification. With cadaver donors, an agarose gel electrophoresis is necessary to assure amplification. The amplicon (2 ul) is placed in each of the 28 wells, dried at 60°C for 10 minutes, and denatured. Hybridization is accomplished with the probe sets at 46°C, followed by several washes. The conjugate is added, followed by color development. The resulting black dots are scored and allelic patterns resolved. Twelve cadaver donors were DNA typed by the Reverse Dot Blot Procedure for DRB1,3,4,5, developed and proven accurate by our laboratory, and with the SSOP kit. Concordance was 100% between the two methods. There were eleven heterozygotes and one homozygote. Eleven of twelve gave group-specific resolution with both tests, while one individual gave high resolution with the SSOP. In summary, the SSOP kit is facile, rapid, and allows typing of cadaveric donors in a timely fashion.
C-7.4 #198
THE USE OF DRIED BLOOD STAINS AS A N A R C H I V A B L E SPECIMEN FOR CLASS II HLA TYPING BY THE POLYMERASE CHAIN REACTION. U Heine, E. Owens, J. Medley, K. VonCannon, and JM Mason, Department of HLA/Paternity, Roche Biomedical Laboratories, Burlington, NC. Dried blood stains ("DBS")were e v a l u a t e d as potential specimens for DNA typing by PCR. The stability of such specimens over time was evaluated in two independent studies. First, 27 month old DBS were a m p l i f i e d with primers to the p o l y m o r p h i s m at DISS0. These specimens had been subjected to this assay as part of a p a t e r n i t y test panel in 1991. These specimens were then typed for HLA DRB and DQB by PCR/SSOP. All 22 specimens in this study were suitable for typing at all three loci. Second, Fresh blood was collected from twenty u n r e l a t e d individuals into N a : H e p a r i n tubes and typed for DRB by standard serology. DBS were then prepared and immediately typed for DRB by PCR. The PCR assay was repeated p e r i o d i c a l l y over time as part of an ongoing 5 year study. Only two discrepancies were noted in the entire study, and these were between serological and DNA typing. DBS were found to be stable at room temperature for the entire period of this study and p r o v i d e d reproducible results with all anticoagulants evaluated (Na:Heparin, ACD, and EDTA)