One‐Step Quantitative Single Nucleotide Polymorphism (SNP) Diagnosis By Modified Loop‐Mediated Isothermal Amplification (mLAMP)

Abstract

Single nucleotide polymorphism (SNP) detection usually needs two steps: amplification (mostly by polymerase chain reaction) and genotyping of SNP by using the amplification products. To shorten the time and simplify the detection, it is ideal to develop a one-step method, in which the amplification itself can be the SNP detection signal. Meanwhile the difficulty in developing one-step technology is the suppression of the background amplification. In this work, we designed an artificial mismatch into the third position from the 3' terminus of the probes to prevent false positive extension reaction of the probes, which greatly improved the selectivity of mutation detection. We integrated this high selective version of primer extension reaction with a rapid and isothermal nucleic acid amplification loop-mediated isothermal amplification (LAMP) method to distinguish between wild-type and mutant DNA. SYBR ® Green I is selected as the fluorescent dye that is tested for real-time measurement of the LAMP reaction by its fluorescence intensity.100aM single strand DNA (ssDNA) or double strand DNA (dsDNA) targets can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LAMP and gives a wide dynamic range for detection of DNA targets (from 100aM to 1 pM).Single nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, which are single nucleotide change with an estimated frequency of one to two polymorphic nucleotides per kilobase. [1] They can be used as not only high-resolution genetic markers for mapping gene, defining population structure, and performing gene association studies but also a fundamental tool for drug discovery and identification of numerous genetic diseases. [2] SNP detection generally needs two steps: amplification of target DNA and genotyping of SNP by using the amplification products. [3] Amplification, usually by the polymerase chain reaction (PCR), is a powerful tool to achieve enough sensitivity for detection of genomic DNA. In principle, the allele-specific PCR, by designing a primer with the nucleotide at its 3'-end complementary to the base at the SNP site in the detected targets, can directly detect SNP. However, the DNA polymerase cannot provide enough specificity to accurately discriminate single-base mis-match. The mismatched primer extension often occurs and the extension products can subsequently be exponentially amplified with PCR prone to generate of false positive results. [4] Therefore, the genomic DNA region that spans the SNP sites of interest needed to be amplified by PCR, and then the amplification products were genotyped at the SNP sites by using allele-specific hybridization, oligonucleic ligation, [5] invasive cleavage, [6] primer extension, and so on, [7] The padlock probes can be specifically ligated with DNA ligase when they are perfectly complementary to the DN...

Single-nucleotide polymorphism-based epidemiological analysis of KoreanMycobacterium bovisisolates

“…LAMP-based technologies have successfully achieved accurate and fast SNP analysis. In addition, allele-specific LAMP, one-step strand displacement-coupled LAMP, and LAMP combined with allele-selective oligonucleotide hybridization have been applied to SNP analysis [ 39 ]. The combination of these and current SNP typing methods would enable extremely fast and accurate SNP typing of M. bovis and other important pathogens.…”

Electrochemical biosensors for analysis of DNA point mutations in cancer research

“…5), and thus not many EC assays used LAMP for point mutation detection (Table 2). In fact, most assays were fluorimetric or colorimetric, targeting, e.g., mutations in TP53, KRAS, BRAF, or EGFR genes [129,130]. An exception is a work by Wang et al that used a glucometer for analysis of V600E BRAF mutation, where the V600E mutation was localized within the F loop [80].…”