Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

Abstract

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS)

“…S9 † for PAGE analysis of the PCR product). 64 The resulting PCR mixture containing 71-mer singlestranded DNA (Fig. 6A), which is an identical sequence to the targeted mRNA region, was directly assessed using a CHOMS reaction without further manipulations.…”

<p>Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes</p>

“…In previous studies, LAMP assays were used to detect the F167Y mutation in Fusarium graminearum and KRAS mutation patients with cancer. 16,17 Additionally, LAMP assays were used to detect the haptoglobin gene deletion in alkaline-denatured blood. 18 In this study, we found that the target sequence was successfully amplified and that the wild type and target could be distinguished.…”

Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: A rapid on‐site detection of gene doping

“…[41] Mutation responsible for cancer development can also be detected by this methodology. [42] In a different context, Centeno-Cuadros et al developed a LAMP protocol for sex determination of raptor species. [43] In comparison with PCR-based methods, LAMP amplification demonstrates better sensitivity and specificity.…”