DNA melting analysis.

Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon.

Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.

Aims: The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (HS). In this study, we assessed the role of CBS in HS biogenesis.

Results: We show that despite discouraging enzyme kinetics of alternative HS-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of HS.

Heterozygote PCR product melting curve prediction.

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes.

Quantum method for fluorescence background removal in DNA melting analysis.

Fluorescent high-resolution DNA melting analysis is a robust method of genotyping and mutation scanning. However, removing background fluorescence is important for accurate classification and to correctly display helicity. Linear baseline extrapolation, commonly used with absorbance, often fails at low temperatures when fluorescence is used.

Purification and characterization of the wild type and truncated human cystathionine beta-synthase enzymes expressed in E. coli.

In this paper, we describe the expression and characterization of recombinant human cystathionine beta-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the N-terminus.

Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.

Background: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping.

Methods: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions.

High-resolution DNA melting analysis for simple and efficient molecular diagnostics.

High-resolution melting of DNA is a simple solution for genotyping, mutation scanning and sequence matching. The melting profile of a PCR product depends on its GC content, length, sequence and heterozygosity and is best monitored with saturating dyes that fluoresce in the presence of double-stranded DNA. Genotyping of most variants is possible by the melting temperature of the PCR products, while all variants can be genotyped with unlabeled probes.