Genotyping accuracy of high-resolution DNA melting instruments

pre-printHigh-resolution DNA melting is a closed-tube method for genotyping and variant scanning that depends on the thermal stability of PCR-generated products. Instruments vary in thermal precision, sample format, melting rates, acquisition, and software. Instrument genotyping accuracy has not been assessed. Each genotype of the single nucleotide variant (SNV) (c.3405-29A>T) of CPS1 (carbamoyl-phosphate synthase 1, mitochondrial) was amplified by PCR in the presence of LCGreen Plus with 4 PCR product lengths. After blinding and genotype randomization, samples were melted in 10 instrument configurations under conditions recommended by the manufacturer. For each configuration and PCR product length, we analyzed 32-96 samples (depending on batch size) with both commercial and custom software. We assessed the accuracy of heterozygote detection and homozygote differentiation of a difficult, nearest- neighbor symmetric, class 4 variant with predicted <Tmof 0.00 °C

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments

pre-printBACKGROUND: Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. METHODS: A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. RESULTS: The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. CONCLUSIONS: Simple DNAextension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations,DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR

Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise ∼16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42–86 bp) genotyping on the LightScanner®. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average Tm standard deviations of these targets decreased from 0.067°C before calibration to 0.022°C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086°C to 0.032°C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by Tm even though current nearest neighbor models predict that the two homozygous alleles would be identical

Human kallikrein gene 13 (KLK13) expression by quantitative RT–PCR: an independent indicator of favourable prognosis in breast cancer

Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase–polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival (P<0.001 and P=0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I–II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 (P=0.001) and 0.24 (P=0.008), respectively, for the Grade I–II group; 0.36 (P=0.008) and 0.44 (P=0.038), respectively, for the node positive group and 0.36 (P=0.008) and 0.18 (P=0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 (P=0.012). For patients in the node positive and oestrogen receptor positive subgroup (n=51) the adjusted hazard ratio was 0.25 (P=0.006) and for the node positive and progesterone receptor positive subgroup (n=46) the hazard ratio was 0.24 (P=0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase–polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Assay, Purification, and Characterization of a Pantetheine Hydrolyzing Enzyme from Pig Kidney

A microsomal glycoprotein hydrolyzing pantetheine to pantothenate and cysteamine has been solubilized and purified to homogeneity as determined by sodium dodecylsulfate electrophoresis. Four rapid, independent assays of pantetheine hydrolysis are described and compared along with a method for localizing enzymatic activity on polyacrylamide gels. The enzyme is solubilized on exposure to butanol and purified by heat treatment, (NH4)2SO4 fractionation, hydrophobic chromatography, and hydroxyapatite chromatography. The glycoprotein, purified 5600-fold in 22% yield, has a specific activity of 14 μmoles pantothenate produced/min/mg of protein, 35 times that previously reported. The enzyme has a pH optimum of 9.0-9.5 and a Km of 20 μM for D-pantetheine. Its molecular weight has been estimated by gel filtration (54,000) and sodium dodecylsulfate electrophoresis (60,000). Amino acid analysis indicates a high content of isoleucine (11.3 mole %) and phenylalanine (7.4 mole %) with an absorbancy index El%cm (280 nm) of 11 .3. Galactose, mannose, fucose, and glucose are present in decreasing concentrations totaling 90 μg of neutral hexoses per mg of protein. Galactosamine and sialic acid are also present. The enzymatic hydrolysis of various pantetheine analogs indicate the enzyme 1s specificity is high for the pantothenate moiety but low for the cysteamine portion. The enzyme should be useful in routine 11 total 11 pantothenate determinations in tissues

MIQE: A Step Toward More Robust and Reproducible Quantitative PCR

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