Journal: Nucleic Acids Research

Article Title: MLGA—a rapid and cost-efficient assay for gene copy-number analysis

doi: 10.1093/nar/gkm651

Figure Lengend Snippet: ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.

Article Snippet: Pooled samples of male and female DNA from Promega (cat# G147A 20745001, cat# G152 20215001) were used as references.

Techniques: Multiplex Assay, Ligation, Amplification, Plasmid Preparation, Hybridization, Sequencing, Electrophoresis

Journal: Clinical chemistry and laboratory medicine

Article Title: Rapid Screening for Targeted Genetic Variants via High-Resolution Melting Curve Analysis

doi: 10.1515/cclm-2016-0603

Figure Lengend Snippet: HRM curves distinguish genotypes. For each SNP, representative HRM curves of reference (black curves), heterozygous (green curves), and homozygous (red curves) DNA are illustrated. Single nucleotide base pair mutations caused a shift in melting temperature of the target amplicon, allowing for visual separation of genotypes with HRM curves. No homozygous control DNA template was available for CYP3A5*2 (F).

Article Snippet: DNA templates Reference sequence, heterozygous, and homozygous control template DNA samples containing each SNP of interest were obtained from the Coriell Institute for Medical Research (Camden, NJ, USA) after obtaining proper institutional approval.

Techniques: Amplification, Control

Journal: Clinical chemistry and laboratory medicine

Article Title: Rapid Screening for Targeted Genetic Variants via High-Resolution Melting Curve Analysis

doi: 10.1515/cclm-2016-0603

Figure Lengend Snippet: HRM difference plots. Differences in melting curve shape were visualized by subtracting the curves from a reference sequence control curve. Normalized fluorescence minus reference (y-axis) was plotted against temperature (x-axis) for each SNP. Heterozygous (green curves) and homozygous (red curves) samples were clearly delineated from reference samples (black curves). With the described HRM screening approach, both heterozygous and homozygous samples would be collectively called as ‘variant.’

Article Snippet: DNA templates Reference sequence, heterozygous, and homozygous control template DNA samples containing each SNP of interest were obtained from the Coriell Institute for Medical Research (Camden, NJ, USA) after obtaining proper institutional approval.

Techniques: Sequencing, Control, Fluorescence, Variant Assay

Journal: Clinical chemistry and laboratory medicine

Article Title: Rapid Screening for Targeted Genetic Variants via High-Resolution Melting Curve Analysis

doi: 10.1515/cclm-2016-0603

Figure Lengend Snippet: Concordance with Sanger sequencing. A representative example is shown for CYP3A5*3, a substitution of thymine for cytosine. With Sanger sequencing, reference samples displayed thymine at the SNP location (A), homozygous samples displayed cytosine (C), and heterozygous samples displayed peaks for both alleles (B). HRM difference plots show the same unknown samples (cyan curves) alongside DNA controls of known genotype (D–F).

Article Snippet: DNA templates Reference sequence, heterozygous, and homozygous control template DNA samples containing each SNP of interest were obtained from the Coriell Institute for Medical Research (Camden, NJ, USA) after obtaining proper institutional approval.

Techniques: Sequencing