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

Objective: The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable.

Methods: The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed.

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

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses.

TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.

Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR.

Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations.

The data in this article are related to the research article entitled "Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations" Botezatu et al. [1]. Somatic mutations in the PIK3CA gene ("hot spots" in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy.

Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations.

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS.

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the "closed tube" format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the "open tube" format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed.

Tube gel isotachophoresis: a method for quantitative isolation of nucleic acids from diluted solutions.

The technique of isotachophoresis is intended for separation of molecules having different electrophoretic mobilities in a nonhomogeneous electric field. Since the mobility of nucleic acids in water solutions is uniform and does not depend on their size (because of a uniform distribution of negatively charged phosphate groups along the molecule), isotachophoresis will concentrate rather than separate them in the mobile borderline zone between the rapid (Cl(-)) and the slow (β-alanine(-)) anions. This idea served as the basis for elaboration of a novel method for isolation of nucleic acids from diluted solutions.

Transrenal DNA as a diagnostic tool: important technical notes.

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA.