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

Abstract

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%. Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Cationic conjugated polymers (CCPs) with a large number of repeated absorbing units can transfer the excitation energy along the whole CCP backbone to the chromophore reporter [12]. Because of their unique light-harvesting properties, CCPs provide an effective platform for homogeneous SNP detection based on the formation of polyelectrolyte complexes stabilized by electrostatic interactions between the CCPs and negatively charged DNA. When a fluorophore-labeled nucleotide is incorporated into the DNA chain during the primer extension reaction at an SNP

Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing

“…Recently, RCA had been utilized to study and develop sensitive detection methods for DNA [ 9 , 11 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ], RNA [ 25 , 26 , 27 ], DNA methylation [ 28 , 29 ], single nucleotide polymorphisms (SNP) [ 30 , 31 , 32 ], small molecules [ 7 , 33 , 34 ], target proteins [ 10 , 35 ], and cancer cells [ 6 , 36 , 37 ]. The circular templates are designed in RCA so that a single binding event can be amplified over a thousand-fold.…”

Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA

“…Fluorescent detection has the advantage that multiple RNA/DNA species can be detected simultaneously using spectrally separable dyes [8]. A number of fluorescent-labeled nucleotides have also been tested for direct incorporation during RCA and direct or indirect detection by fluorescence resonance energy transfer (FRET) [25,26]. However, most of these detection methods for RCA products have relatively low sensitivity (e.g., only one or a few labels are usually present in the detection probes), high background due to non-specific random hybridization and to the presence of endogenous high molecular weight DNA, and are time-consuming due to the long time to hybridize and wash the non-specifically bound probes from the sample.…”

A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing-Loss Detection

“…SNPs (single nucleotide polymorphisms) and InDels (insertion-deletion markers), the most common heritable variations in the human genome, have been extensively applied to the analysis of disease genetics, population genetics, forensic medicine, pharmacogenomics, and biodiversity as the third generation of genetic markers, and they will continue to play important roles in the development of life sciences. − To facilitate relevant research studies, many methods were developed for SNPs/InDels detection, which can be generally divided into two types: (1) The gel-based methods with low throughput, including restriction enzyme fragment length polymorphic analysis (RFLP), single-strand conformational polymorphic analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), conformation sensitive gel electrophoresis (CSGE), oligonucleotide link analysis (OLA), and allele-specific polymerase chain reaction analysis (ASP). These methods have the advantages of simple equipment requirements and low costs but are limited by their tedious operations, long analysis periods, and low throughputs. − (2) The high throughput methods follow the principle of allele-specific site hybridization (ASH), mass spectrometry (MS), , denaturing high performance liquid chromatography (DHPLC), allele-specific site primer extension (ASPE), single base extension (SBCE), or target sequencing. − For instance, the SNaPshot − and TaqMan OpenArray systems − have been used for high-throughput screening of SNPs, , while the GoldenGate platform can detect 1536 SNPs at one time. These methods have significantly improved throughputs but rely highly on costly instruments and need many samples to decrease the cost for each sample.…”