3′-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction
10.3321/j.issn:0258-879X.2003.04.014
- VernacularTitle:3′末端碱基游移混合引物提高多变区基因片段PCR检测的阳性率
- Author:
Wen-Sheng XU
1
;
Xiao-Hui MIAO
;
Wen-Ya WU
;
Yong HAO
Author Information
1. Changzheng Hospital Second Military Medical University
- Keywords:
primers;
mismatch;
polymerase chain reaction;
false negative
- From:
Academic Journal of Second Military Medical University
2003;24(4):399-402
- CountryChina
- Language:Chinese
-
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.
