A Stambergova

Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner.

Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High‐resolution melting curve (HRM) analysis is a promising and economical method for high‐throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and high‐standard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScannerTM (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1‐specific mutation screening test using the 96‐well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient‐derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs). Hum Mutat 30:1–11, 2009.

Diagnostic method validation: High resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene.

OBJECTIVES According to OECD guidelines methods implemented in a diagnostic laboratory should be properly validated prior their implementation. For this purpose we selected genotyping by High Resolution Melting (HRM) of small amplicons using common variants in MTHFR as a model. DESIGN AND METHODS We selected previously typed samples on which selected analytical validation-related parameters relevant to DNA diagnostics - specificity, sensitivity, precision, robustness and ability to perform reliable calls were evaluated. RESULTS Correct genotype was assigned in 375/381 (98.4%) for c.677 C>T (rs1801133: C>T; p.A222 V) and in 102/104 (98.1%) for c.1298 A>C (rs1801131: A>C; p.E429A) of all cases. Low analytical failure rate and very high specificity/sensitivity were achieved. Similarly, precision and robustness were consistent. CONCLUSIONS We have successfully validated HRM of small amplicons using common MTHFR variants as a model. We proved that this technique is highly reliable for routine diagnostics and our diagnostic validation strategy can serve as a model for other applications.

Affinity Chromatography with Immobilized Benzeneboronates

Boronate affinity chromatography is based on the unique ability of ionized boronic acids to reversibly form cyclic (five- or six-membered ring) esters with diol groups under mild conditions. An aqueous medium and a 1,2-diol in a cis-coplanar geometry (Fig. 1) or a 1,3-diol of proper geometry are required for this interaction. Such diol functions are present in many biologically significant structures, particularly saccharides, and, therefore, boronate chromatography occupies an important position in analytical and preparative chromatography of these substances. Among the advantages of boronate chromatography, in comparison with many other affinity methods, are the availability and stability of the ligands. Boronate chromatography has already been subject of several reviews (Bergold and Scouten, 1983; Carlsohn and Hartmann, 1979; Dean et al., 1983; Fulton, 1981; Mazzeo and Krull, 1989). We have, therefore, restricted this chapter to a discussion of the basic principles of the process and a description of developments over the past few years (chiefly since 1983).