Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock pr...

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Bibliographic Details
Published in:Chinese science bulletin 2011-11, Vol.56 (31), p.3247-3252, Article 3247
Main Authors: Tang, ZhiYuan, Cheng, YongQiang, Du, Qing, Zhang, HongXia, Li, ZhengPing
Format: Article
Language:English
Subjects:
base pair mismatch
Chemistry/Food Science
DNA-directed DNA polymerase
DNA序列
Earth Sciences
electrostatic interactions
Engineering
fluorescein
gene frequency
Humanities and Social Sciences
Life Sciences
multidisciplinary
mutants
Physics
Science
Science (multidisciplinary)
single nucleotide polymorphism
single-stranded DNA
一体化
共轭聚合物
单核苷酸多态性
同质化
多态性检测
滚环
阳离子
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Summary:A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.
ISSN:1001-6538
1861-9541
DOI:10.1007/s11434-011-4663-0