Improvement of HLA class I and class II PCR-SSP typing by using timed-release activity of DNA polymerase.
Variable amounts of non-specific amplification may occur in HLA PCR-SSP typing, and this can be significantly reduced by the use of AmpliTaq Gold. In an effort to achieve an optimal balance between specificity and efficiency of the PCR amplification for both HLA alleles and the internal control, we designed a system of timed-release activity by combining two different Taq DNA polymerases. The reaction was started at a relatively low level of enzyme activity and as thermal cycling progressed more and more Ampli-Taq Gold was slowly activated for the reaction to continue. We applied this system to all routine HLA PCR-SSP typing. The number of repeat typings due to non-specific amplification and/or amplification failure of the internal control was remarkably reduced.
Chen
D F
DF
Department for Bloodbank, Immunohematology and Transfusion Medicine, Hannover Medical University, Germany. dfchen@cbr.med.harvard.edu
Seibert
I
I
Chen
H Y
HY
Herbst-Kiene
I
I
Pastucha
L T
LT
Stangel
W
W
D017931:DNA Primers; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000235:genetics*; D000949:Histocompatibility Antigens Class II / Q000145:classification / Q000235:genetics*; D006650:Histocompatibility Testing; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase / Q000378:metabolism*; D013997:Time Factors
D017931:DNA Primers; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase