scholarly journals Association between the p73 gene G4C14-to-A4T14 single nucleotide polymorphism and risk of cervical cancer by high resolution melting and PCR with confronting two-pair primers in a Chinese population

2016 ◽  
Vol 12 (1) ◽  
pp. 721-726 ◽  
Author(s):  
HAIYAN GUO ◽  
SHAODI YANG ◽  
LIJIAN XU ◽  
DING LI ◽  
JIANXIN TANG ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Chinese Population ◽  
High Resolution Melting ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
P73 Gene

scholarly journals Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

2004 ◽  
Vol 50 (10) ◽  
pp. 1748-1754 ◽  
Author(s):  
Gudrun H Reed ◽  
Carl T Wittwer
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Sensitivity And Specificity ◽  
High Resolution Melting ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Product Size ◽  
Pcr Products ◽  
Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

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