Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Ботезату, И. В.; Кондратова, В. Н.; Шелепов, В. П.; Мазуренко, Н. Н.; Цыганова, И В; Susova, Olga Yu.; Lichtenstein, A. V.

doi:10.1016/j.ab.2019.113517

Cited by 12 publications

(6 citation statements)

References 37 publications

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“…Moreover, the test involves many washing and incubation steps with a total analysis time of about 2 h [ 48 ]. Compared to the previously reported biosensors for ctDNA detection in real samples ( Table S3 ) [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ], the advantages of the proposed multi-plex test are related mostly to the cost, run time, and simplicity, as well as the universality and advanced multiplexing potential.…”

Section: Discussionmentioning

confidence: 92%

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

et al. 2022

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In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

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Section: Discussionmentioning

confidence: 92%

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

et al. 2022

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“…Experimental and clinical studies typically require evaluation of the copy number of a target gene and the presence of polymorphisms, particularly mutations. Quantitative real time PCR with TaqMan probes is often used to evaluate copy number, and post-PCR DMA to detect polymorphisms 8,9,18 . A disadvantage of this approach is that the quantitative real time PCR is symmetric, while DMA with TaqMan probes requires an asymmetric PCR; therefore, two independent specific reactions are necessary.…”

Section: Resultsmentioning

confidence: 99%

“…5). www.nature.com/scientificreports www.nature.com/scientificreports/ The DMA assay permits detection of ~5% of mutant alleles in wild-type DNA 9 . A range of techniques are used for mutant enrichment during PCR.…”

Section: Products Of Pcr Amplification the Comparison Of Possible Stmentioning

confidence: 99%

“…Since this effect is more pronounced in the case of perfect hybrids, compared to mismatched ones, the TaqMan probes can be used for mutant enrichment. If relatively minor variances in the stability of the probe/template complexes do not give a noticeable effect under standard PCR conditions, then under suboptimal PCR conditions (for example, short PCR cycles), a strong effect of mutant enrichment and many-fold increase in sensitivity can be achieved 9,10,19 .…”

Section: Products Of Pcr Amplification the Comparison Of Possible Stmentioning

confidence: 99%

“…Mutant-enriched SLAM-MS. PCR conditions were as described above except that after primary DNA denaturation (5 min at 95 °C), a two-stage PCR was performed: 50 short cycles ( Quantitation of DnA melting analysis. Quantitation of DNA melt peaks was carried out as described earlier 9 . We assume that the number ratio of mutant and wild-type alleles has a linear relationship with the area ratio of respective melting peaks.…”

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SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

Kondratova

Botezatu

Shelepov

et al. 2020

Sci Rep

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DnA Melting Analysis (DMA) with a taqMan probe covering the mutation "hot spot" is a simple, sensitive, and "closed tube" method of mutation detection. However, DMA requires asymmetric pcR to produce single-stranded amplicons capable of interacting with taqMan probes. this makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent pcRs. the SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with taqMan probes. Hybrids of stem-loops and complementary taqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. in addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. for mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay. In basic cancer research, priority is given to large-scale (genome-and exome-wide) next generation sequencing (NGS) methods, which give a panoramic picture of defects in the cancer genome. However, for clinical diagnostics, a "targeted approach" is more often required, i.e. screening of previously known clinically important mutations, in particular in the KRAS, NRAS, BRAF, EGFR, and PIK3CA genes 1-7. In this case, the priorities are the feasibility of the method in a clinical laboratory, and its simplicity, performance, and sensitivity. These requirements are largely met by asymmetric PCR with a TaqMan probe covering the mutation "hot spot" and subsequent DMA (DNA Melting Analysis), which is one of the most simple, fast, and least costly methods to detect DNA mutations. Moreover, it is implemented in the "closed tube format" that avoids any additional manipulations and eliminates sample cross-contamination 8,9. The short length of the TaqMan probes (20-30 nucleotides) facilitates strong mismatch-induced changes in melting curves and clear discrimination of wild-type and mutant melt peaks. Computation of the areas under the peaks using specialized software permits quantification of the ratio of wild and mutant alleles 9. Furthermore, under special (suboptimal) PCR conditions, the TaqMan probes serve as blocking agents contributing to the enriched synthesis of mutant alleles 10. An inherent drawback of this method is the asymmetric mode of PCR necessary for accumulation of single-stranded amplicons ("targets" for TaqMan probes). This leads to a number of limitations: (i) a decrease in the amplification efficiency, ...

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Aberrant methylation scanning by quantitative DNA melting analysis with hybridization probes as exemplified by liquid biopsy of SEPT9 and HIST1H4F in colorectal cancer

Botezatu,

Kondratova,

Stroganova

et al. 2023

Clinica Chimica Acta

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Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Cited by 12 publications

References 37 publications

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

Aberrant methylation scanning by quantitative DNA melting analysis with hybridization probes as exemplified by liquid biopsy of SEPT9 and HIST1H4F in colorectal cancer

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