scholarly journals Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays

2003 ◽  
Vol 31 (3) ◽  
pp. 7e-7 ◽  
Author(s):  
D. Di Giusto
Keyword(s):  
Single Base Extension ◽  
Single Base ◽  
Single Substrate ◽  
Base Extension

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping

2010 ◽  
Vol 163 (5) ◽  
pp. 573-576 ◽  
Author(s):  
Guibo Liu ◽  
Yongxia Cheng ◽  
Wei Zhao ◽  
Zaishun Jin ◽  
Hongbo Shan ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Single Base Extension ◽  
Single Nucleotide ◽  
Single Base ◽  
Base Extension

In-Situ Mutation Detection by Magnetic Beads-Probe Based on Single Base Extension and Its Application in Genotyping of Hepatitis B Virus Pre-C Region 1896nt Locus Single Nucleotide Polymorphisms

2019 ◽  
Vol 15 (12) ◽  
pp. 2393-2400
Author(s):  
Xianbo Mou ◽  
Danni Sheng ◽  
Zhu Chen ◽  
Mei Liu ◽  
Yuan Liu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Fluorescence Intensity ◽  
Magnetic Beads ◽  
Mutation Detection ◽  
Single Base Extension ◽  
Single Base ◽  
Base Extension ◽  
B Virus

Hepatitis B virus (HBV) is closely related to occurrence and development of viral hepatitis. A mutation of 1896nt locus in its pre-C region can promote replication of HBV DNA and improve stability of pre-genome RNA structure, and can even help HBV evade immune clearance. In this study, magnetic beads-probe (MBs@probe) method, combined with single base extension (SBE) technology, was developed for in-situ mutation detection of HBV pre-C region 1896nt locus. Before successfully completing the genotyping of 165 HBV samples, the crucial reaction conditions were first optimized, such as SBE temperature, MBs size and amount, and probe concentration on the surface of MBs. Experimental results showed that these conditions had significant effects on MBs@probe in-situ mutation detection. Comprehensive considerations, such as 58 °C of SBE temperature, high fluorescence intensity and signal-to-noise ratios (SNRs) were obtained when MBs@probe complex was made by 100 μg of 300 nm-MBs and 3.0 μM of probes in the system. Finally, 1896nt locus mutation in pre-C region of 165 HBV samples was successfully genotyped, among which 71 HBV samples were wild types and the remaining 94 samples were mutant types. Meanwhile, 14 randomly chosen samples were taken to further analyze fluorescence intensity and SNRs respectively, and sequencing results for the first two samples were consistent with results from the MBs@probe in-situ mutation detection method. Compared with two-color fluorescence hybridization (TCFH) genotyping technology, this method generally improves the SNRs to more than 10 (which is more than 2-fold), has higher reliability and is more suitable to detect SNPs for known sites.

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