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Non-radioactive magnetic bead hybridization: A practical test for clinical HLA-DNA oligotyping
Abstracts
99
C 7.4.185
PCR-BASED VNTR ANALYSIS OF CHIMERISM IN BONE MARROW TRANSPLANT PATIENTS. AG Smith. C McFarland, M Andreani, E Mickelson, PJ Martin and JA Hansen, F Hutchinson Cancer Research Center, Seattle WA. The analysis of chimerism in bone marrow transplant (BMT) patients requires techniques capable of detecting polymorphic markers on multiple chromosomes (C) in both patient and donor. In our laboratory, this historically has been accomplished by restriction fragment length polymorphism (RFLP) analysis of variable numbers of tandem repeat (VNTR) loci. Recently, primers have been described for the polymerase chain reaction (PCR) amplification of several VNTR loci: D1S80 on C1, apoB on C2 and pYNZ22 on C17. To determine the efficacy of the PCR-based method in documenting marrow engraftment status, we compared the results of RFLP vs. PCR analysis in 43 post-BMT cases. Twenty-five of the patient-donor pairs were HLA identical siblings (SIB), while 18 were unrelated (URD) pairs. With the RFLP technique using 3 VNTR probes, informative DNA markers were detected in 92% of the SIB and 100% of the URD cases. With the PCR-based D1S80 assay alone, informative markers were detected in 39% of SIB and 72% of URD cases; addition of the apoB assay increased the detection to 66% and 89%, respectively. Preliminary data indicate that addition of the pYNZ22 assay to the D1S80 and apoB assays increases the power of discrimination to that obtainable with RFLP. In addition, the PCR method allows quantification of as little as 5% donor or patient DNA in a heterogeneous sample, can be performed in one day, and is cost effective. These results indicate the utility and advantage of PCRbased chimerism assays in clinical BMT.
C 7.4.186
NON-RADIOACTIVE
MAGNETIC
BEAD
HYBRIDIZATION:
A
PRACTICAL
TEST
FOR
CLINICAL HLA-DNA OLIGOTYPING. A Lazaro, M Fernandez-Vina, Z Liu and P Stastny, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas. The use of PCR and oligonucleotide hybridization has greatly increased the accuracy and resolution of typing of HLA-Class II alleles. However, current methods are done in batches and take too long. We have developed a method using enzyme-linked oligonucleotide probes and immobilized PCR products on magnetic beads in 96-well trays. Trays preloaded with 5'-amino-group oligonucleotides covalently linked with alkaline phosphatase have been kept at 4 ~ for at least 5 weeks without loss of enzyme-probe activity. DR beta amplification was performed with exon-2 flanking primers. One of them is biotinylated and is used to immobilize the PCR products on streptavidin-magnetic beads. Beads with attached PCR products are dispensed into the preloaded trays and hybridization is allowed to proceed. Excess probes are washed out by inversion of the tray using a magnetic plate to hold the beads in place. Washing solutions are used to remove non-matching probes. Finally, color reactions are developed by a sensitive assay with enzymatic amplification that produces readings in less than 25 min. Typing currently makes use of a set of 19 probes and a monomorphic positive control probe that allows broad typing of all serologically-defined DR alleles. We have tested 20 homozygous B cell lines and 30 samples from heterozygous individuals previously typed by dot blot hybridization. Expected negative readings at 492 nm ranged from OD 0.019 to 0.065; expected positive readings ranged from OD 0.865 to 1.993. Coupled with a quick D N A preparation method, results can be obtained in less than five hours. This method should be easily performed in small laboratories. It is accurate, reproducible, and sensitive and will make oligotyping for HLA alleles more convenient for testing clinical samples.
99
C 7.4.185
PCR-BASED VNTR ANALYSIS OF CHIMERISM IN BONE MARROW TRANSPLANT PATIENTS. AG Smith. C McFarland, M Andreani, E Mickelson, PJ Martin and JA Hansen, F Hutchinson Cancer Research Center, Seattle WA. The analysis of chimerism in bone marrow transplant (BMT) patients requires techniques capable of detecting polymorphic markers on multiple chromosomes (C) in both patient and donor. In our laboratory, this historically has been accomplished by restriction fragment length polymorphism (RFLP) analysis of variable numbers of tandem repeat (VNTR) loci. Recently, primers have been described for the polymerase chain reaction (PCR) amplification of several VNTR loci: D1S80 on C1, apoB on C2 and pYNZ22 on C17. To determine the efficacy of the PCR-based method in documenting marrow engraftment status, we compared the results of RFLP vs. PCR analysis in 43 post-BMT cases. Twenty-five of the patient-donor pairs were HLA identical siblings (SIB), while 18 were unrelated (URD) pairs. With the RFLP technique using 3 VNTR probes, informative DNA markers were detected in 92% of the SIB and 100% of the URD cases. With the PCR-based D1S80 assay alone, informative markers were detected in 39% of SIB and 72% of URD cases; addition of the apoB assay increased the detection to 66% and 89%, respectively. Preliminary data indicate that addition of the pYNZ22 assay to the D1S80 and apoB assays increases the power of discrimination to that obtainable with RFLP. In addition, the PCR method allows quantification of as little as 5% donor or patient DNA in a heterogeneous sample, can be performed in one day, and is cost effective. These results indicate the utility and advantage of PCRbased chimerism assays in clinical BMT.
C 7.4.186
NON-RADIOACTIVE
MAGNETIC
BEAD
HYBRIDIZATION:
A
PRACTICAL
TEST
FOR
CLINICAL HLA-DNA OLIGOTYPING. A Lazaro, M Fernandez-Vina, Z Liu and P Stastny, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas. The use of PCR and oligonucleotide hybridization has greatly increased the accuracy and resolution of typing of HLA-Class II alleles. However, current methods are done in batches and take too long. We have developed a method using enzyme-linked oligonucleotide probes and immobilized PCR products on magnetic beads in 96-well trays. Trays preloaded with 5'-amino-group oligonucleotides covalently linked with alkaline phosphatase have been kept at 4 ~ for at least 5 weeks without loss of enzyme-probe activity. DR beta amplification was performed with exon-2 flanking primers. One of them is biotinylated and is used to immobilize the PCR products on streptavidin-magnetic beads. Beads with attached PCR products are dispensed into the preloaded trays and hybridization is allowed to proceed. Excess probes are washed out by inversion of the tray using a magnetic plate to hold the beads in place. Washing solutions are used to remove non-matching probes. Finally, color reactions are developed by a sensitive assay with enzymatic amplification that produces readings in less than 25 min. Typing currently makes use of a set of 19 probes and a monomorphic positive control probe that allows broad typing of all serologically-defined DR alleles. We have tested 20 homozygous B cell lines and 30 samples from heterozygous individuals previously typed by dot blot hybridization. Expected negative readings at 492 nm ranged from OD 0.019 to 0.065; expected positive readings ranged from OD 0.865 to 1.993. Coupled with a quick D N A preparation method, results can be obtained in less than five hours. This method should be easily performed in small laboratories. It is accurate, reproducible, and sensitive and will make oligotyping for HLA alleles more convenient for testing clinical samples.