Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

Using cell-free DNA for HCC surveillance and prognosis.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. Its incidence is rising faster than any other cancer in the United States and it remains one of the leading causes of cancer-related deaths worldwide. While advances in massive parallel sequencing and integration of 'omics information have transformed the field of oncology, tissue access is often limited in HCC and a single biopsy is poorly representative of the known genetic heterogeneity of tumours. Liquid biopsy has emerged as a promising strategy for analysing circulating tumour components including circulating tumour DNA. Cell-free DNA and tumour DNA are derived from necrotic, apoptotic and living eukaryotic cells. The profiling of genetic and epigenetic alterations in circulating cell-free DNA has potential clinical applications including early disease detection, prediction of treatment response and prognostication in real time. Novel biomarker candidates for disease detection and monitoring are under study. Of these, methylation analyses of circulating tumour DNA have shown promising performance for early HCC detection in at-risk patients. Assessments of assay performance in longitudinal validation cohorts are ongoing. Implementation of liquid biopsy for HCC will likely improve upon the current surveillance strategy. This review summarises the most recent developments on the role and utility of circulating cell-free DNA in the detection and management of HCC.

The kinetic requirements of extreme qPCR.

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq™), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom "extreme" PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200-500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.