Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

Molecules

Lingwei Wu¹  Quanjun Liu¹  Zhongwei Wu¹ 

[1] ¹State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China ²Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang, 330047, China

关键词: point mutation;    mismatch;    rolling-circle amplification;    T4 DNA ligase;   

DOI  :  10.3390/molecules15020619

来源: mdpi

【 摘 要 】

In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

【 授权许可】

CC BY   
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.