Compromised optimal hybridization temperatures may explain PM plus DQA1 anomalies.
The multiplex DNA typing test kit, PM plus DQA1 relies on 36 different oligonucleotides that all must hybridize correctly for accurate DNA typing. The G + C contents of the probe oligonucleotides range from 47% to 83%. Since G + C content directly affects optimal hybridization temperatures, a wide range of optimal hybridization temperatures is expected. This was verified by two empirically tested formulas. The recommended hybridization temperature is 55 degrees C. The control probe has an estimated optimal temperature of 75 degrees C indicating low stringency of the control. The B allele probe of the GC locus has an estimated optimal hybridization temperature of 70 degrees C. These discrepancies are unexplained by GC clusters or sequence palindromes. The departures from temperature optima may be responsible for observed PM plus DQA1 artifacts and for previously observed, low tolerance of this system to temperature variation. Since optimal temperature and cation concentration are related variables, careful attention to these is recommended as are positive controls demonstrating amplification of all alleles monitored by PM plus DQA1.
Riley
D E
DE
Department of Urology, School of Medicine, University of Washington, Seattle 98195, USA.
D000483:Alleles; D001482:Base Composition; D004247:DNA / Q000032:analysis; D006683:HLA-DQ Antigens / Q000032:analysis*; D059848:HLA-DQ alpha-Chains; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D008285:Major Histocompatibility Complex; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic; D013696:Temperature
D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D011933:Reagent Kits, Diagnostic; D004247:DNA