ABSTRACT
To reduce the risk of 3′-terminal mismatch between primers and template and increase the sensitivity of polymerase chain reaction (PCR) in the detection of variable region of DNA. Methods: A pair of special primer(WU,WD) was designed to amplify a fragment of HBV DNA P gene by PCR. Other 2 similar pairs of primer (MU1, MD1, MU2, MD2) were obtained by knocking off 1 or 2 bases at the 3′-terminal of WU and WD. (1) Special primers (WU, WD) and degeneracy primers(WU, WD, MU1, MU2, MD1, MD2) were used to amplify 27 samples respectively by PCR under the same condition. The sensitivity of each PCR was compared. (2) Using degeneracy primers, serum HBV DNA was amplified from 4 patients who were resistant to lamivudine. The PCR products were sequenced to evaluate the effect of the 3′-terminal mismatch of primers upon PCR. Results: (1) The sensitivity of special primers and degeneracy primers were 70.4%(19/27) and 85.2%(23/27) respectively (P<0.05). (2) The sequencing analysis of the PCR products suggested that the 3′-terminal mismatch of primers caused false negative in the PCR detection. Conclusion: When amplifying the variable region of DNA, the false negative result can be avoided by using 3′-terminus shifted degeneracy primers.
ABSTRACT
To reduce the risk of 3′-terminal mismatch between primers and template and increase the sensitivity of polymerase chain reaction (PCR) in the detection of variable region of DNA. Methods: A pair of special primer(WU,WD) was designed to amplify a fragment of HBV DNA P gene by PCR. Other 2 similar pairs of primer (MU1, MD1, MU2, MD2) were obtained by knocking off 1 or 2 bases at the 3′-terminal of WU and WD. (1) Special primers (WU, WD) and degeneracy primers(WU, WD, MU1, MU2, MD1, MD2) were used to amplify 27 samples respectively by PCR under the same condition. The sensitivity of each PCR was compared. (2) Using degeneracy primers, serum HBV DNA was amplified from 4 patients who were resistant to lamivudine. The PCR products were sequenced to evaluate the effect of the 3′-terminal mismatch of primers upon PCR. Results: (1) The sensitivity of special primers and degeneracy primers were 70.4%(19/27) and 85.2%(23/27) respectively (P<0.05). (2) The sequencing analysis of the PCR products suggested that the 3′-terminal mismatch of primers caused false negative in the PCR detection. Conclusion: When amplifying the variable region of DNA, the false negative result can be avoided by using 3′-terminus shifted degeneracy primers.