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

Abstract

AbstractSingle 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).