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Don't assume contamination, it may be your PCR!
157
Abstracts
7.4 #228
FREQUENCY DATA FOR HLA-DRB4* SUB-TYPES FROM A CAUCASIAN POPULATION. F Jordan, AM Swindell, RH Holman. SA Cleaver and JA Madrigal. The Anthony Nolan Research Centre, The Royal Free Hospital, London. PCR-SSP subtyping for HLA-DRB4* alleles was undertaken on a large panel of the British Caucasoid population (n=238)to ascertain the frequency and linkage disequilibrium of HLA-DRB4* subtypes with their associated HLA-DRBI* (DRBI*04, DRBI*0701, DRBI*090IllI2) and HLADQB 1* alleles. Our findings indicate that within the DRB 1*04 allele group (DRB I *040 1-0409) there is a strong association (> 90%) with DRB4*0103 regardless of the associated HLA-DQB 1* allele. An exception to this may be the HLA-DRB 1*0405 allele which appeared to associate equally with the haplotypes DQBI *020 I IDRB4*0 10 12 and DQBI *0302IDRB4*0103.Within the HLA-DRBI *070 IIDQB 1*0201 halplotype it was observed that there were strong associations with DRB4*0101l (> 60%) and DRB4*0103. DRB4*01012 was not observed to be associated with this haplotype within the population studied. The DRBI *07011DQB 1*0303 haplotype was found to be predominantly associated with DRB4*01012 (>90%) although associations with both DRB4*0101l and DRB4*0103 were ohserved. Finally a strong linkage between DRB I * 09011112 associated DQB I *0303 and DRB4"'OI03 was noted. DRB4*0101 and DRB4*0103 differ by one single amino acid substitution at position 135 in the ~2 domain (1). Although this substitution is not at the peptide binding site, the significance of mismatching at this position and its role in bone marrow transplant outcome needs to be investigated.
1. LA Baxter-Lowe, Eur. J. Immunol. 1994,40,42.
7.4 #229
DON'T ASSUME CONTAMINATION,IT MAY BE YOUR PCR! N. Schroeder, A. Corba, A. Asfour, P. Orchard, S. Vaidya, Department of Pathology, UTMB, Galveston, TX. During our set-up for DNA typing by PCR-SSP we suddenly encountered falsepositive amplifications after many successful typings. The false bands ranged from weak to very strong in consistent reaction tubes. Contamination of isolated DNA, Primers, reagents and the laboratory room and bench tops was eliminated after much lengthy testing. Focus was centered upon the Perkin Elmer Gene Amp 9600 PCR when testing of duplicate samples, ran simultaneously, showed false bands in 1 of the 2 samples. Five different diagnostic tests were run upon the PCR: 1)Heater, 2)Chiller, 3)System Performance, 4)Temperature Calibration Verification and 5)Temperature Uniformity. The PCR passed all five of the tests. The fifth test, Temperature Uniformity, used a two-temperature hold cycle (95 oC and 400C for 2 min.) suggested by the users manual. Using a Perkin Elmer temperature verification system specific to our PRC, readings were taken 90 sec. into each temperature setpoint; these values were registered for 16 different wells spread throughout the sample block. No significant variation was detected. The 95 oC setpoint had a variation of .7oC and the 400C setpoint was 1oC. According to manufacturer's guidelines a variation of >1 oC calls for servicing of the PCR. However, a trend was noticed during the Temperature Uniformity test run. While the block temperature dropped to 400C for a 2 min. hold, the sample temperature dropped anywhere from 38.4oC to 26.4 oC and the time for the sample to stabilize to 39.0oC varied anywhere from 10 sec. to 95 sec. The accuracy of the thermal cycler is one of the most important factors for successful PCR-SSP typing. It is very important to maintain a low thermal gradient «1 oC) across the heating block. Also of critical importance is the annealing temperature of the PCR cycle, a too-low annealing temperature will result in false-positive amplifications. After the PCR was serviced by the manufacturer all false-positive bands were eliminated.
Abstracts
7.4 #228
FREQUENCY DATA FOR HLA-DRB4* SUB-TYPES FROM A CAUCASIAN POPULATION. F Jordan, AM Swindell, RH Holman. SA Cleaver and JA Madrigal. The Anthony Nolan Research Centre, The Royal Free Hospital, London. PCR-SSP subtyping for HLA-DRB4* alleles was undertaken on a large panel of the British Caucasoid population (n=238)to ascertain the frequency and linkage disequilibrium of HLA-DRB4* subtypes with their associated HLA-DRBI* (DRBI*04, DRBI*0701, DRBI*090IllI2) and HLADQB 1* alleles. Our findings indicate that within the DRB 1*04 allele group (DRB I *040 1-0409) there is a strong association (> 90%) with DRB4*0103 regardless of the associated HLA-DQB 1* allele. An exception to this may be the HLA-DRB 1*0405 allele which appeared to associate equally with the haplotypes DQBI *020 I IDRB4*0 10 12 and DQBI *0302IDRB4*0103.Within the HLA-DRBI *070 IIDQB 1*0201 halplotype it was observed that there were strong associations with DRB4*0101l (> 60%) and DRB4*0103. DRB4*01012 was not observed to be associated with this haplotype within the population studied. The DRBI *07011DQB 1*0303 haplotype was found to be predominantly associated with DRB4*01012 (>90%) although associations with both DRB4*0101l and DRB4*0103 were ohserved. Finally a strong linkage between DRB I * 09011112 associated DQB I *0303 and DRB4"'OI03 was noted. DRB4*0101 and DRB4*0103 differ by one single amino acid substitution at position 135 in the ~2 domain (1). Although this substitution is not at the peptide binding site, the significance of mismatching at this position and its role in bone marrow transplant outcome needs to be investigated.
1. LA Baxter-Lowe, Eur. J. Immunol. 1994,40,42.
7.4 #229
DON'T ASSUME CONTAMINATION,IT MAY BE YOUR PCR! N. Schroeder, A. Corba, A. Asfour, P. Orchard, S. Vaidya, Department of Pathology, UTMB, Galveston, TX. During our set-up for DNA typing by PCR-SSP we suddenly encountered falsepositive amplifications after many successful typings. The false bands ranged from weak to very strong in consistent reaction tubes. Contamination of isolated DNA, Primers, reagents and the laboratory room and bench tops was eliminated after much lengthy testing. Focus was centered upon the Perkin Elmer Gene Amp 9600 PCR when testing of duplicate samples, ran simultaneously, showed false bands in 1 of the 2 samples. Five different diagnostic tests were run upon the PCR: 1)Heater, 2)Chiller, 3)System Performance, 4)Temperature Calibration Verification and 5)Temperature Uniformity. The PCR passed all five of the tests. The fifth test, Temperature Uniformity, used a two-temperature hold cycle (95 oC and 400C for 2 min.) suggested by the users manual. Using a Perkin Elmer temperature verification system specific to our PRC, readings were taken 90 sec. into each temperature setpoint; these values were registered for 16 different wells spread throughout the sample block. No significant variation was detected. The 95 oC setpoint had a variation of .7oC and the 400C setpoint was 1oC. According to manufacturer's guidelines a variation of >1 oC calls for servicing of the PCR. However, a trend was noticed during the Temperature Uniformity test run. While the block temperature dropped to 400C for a 2 min. hold, the sample temperature dropped anywhere from 38.4oC to 26.4 oC and the time for the sample to stabilize to 39.0oC varied anywhere from 10 sec. to 95 sec. The accuracy of the thermal cycler is one of the most important factors for successful PCR-SSP typing. It is very important to maintain a low thermal gradient «1 oC) across the heating block. Also of critical importance is the annealing temperature of the PCR cycle, a too-low annealing temperature will result in false-positive amplifications. After the PCR was serviced by the manufacturer all false-positive bands were eliminated.