PCR primers tailed with common sequences (e.g. m13) facilitate DNA sequence reaction set up, enabling any amplicon from any gene to be sequenced using single forward and reverse primers. Mutation scanning techniques such as high

resolution melt curve analysis (HRM) [23] are also amenable to high throughput analysis. Robotic sample processing eliminates inter-user variation in pipetting, resulting in more consistent DNA sequence whilst enhancing sample throughput. Following DNA sequencing, application of sequence analysis software enables rapid identification of variants that differ from the reference sequence (RefSeq). Large deletions and duplications can be identified using multiplex ligation-dependent probe amplification (MLPA) [24,25], micro-array or see more mutation-specific gap-PCR [26]. F8 intra-chromosomal inversions CP-868596 supplier amplified through long or inverse PCR can be analysed using gel electrophoresis

or, for smaller amplicons, by fragment sizing using a DNA sequencer. Previously ‘missing’ deep intronic mutations that affect splicing can be detected by reverse transcription (RT)-PCR from mRNA, gel electrophoresis and DNA sequencing [27,28]. Sequence-variant interpretation software integrates pathogenicity prediction tools for amino acid substitutions and for splice variants along with literature searching for previously reported variants. For amino acid substitutions, these are based on different algorithms for their potential impact on protein structure/function using assessment of physical properties, along 上海皓元医药股份有限公司 with their evolutionary conservation. Mutations potentially affecting splicing are analysed by panels of algorithms analysing presence and relative strength of splice motifs. Locus-specific mutation databases, the Human Gene Mutation Database (HGMD) [29], and the single nucleotide polymorphism database (dbSNP) [30] catalogue and help facilitate pathogenicity interpretation of previously reported variants.

Newly recognized sequence variants underneath PCR amplification primers can be sought with each new release of dbSNP using tools including SNPCheck [31]. Primers can then be redesigned where necessary using online software to prevent mono-allelic amplification resulting in allele dropout and mutations potentially being missed. [32] Reference genetic materials are available for common mutations through the UK National Institute for Biological Standards and Control (NIBSC) [33] and can be used to validate assay performance. LIMS use can facilitate reporting of the results of genetic testing to the referring clinician through ready availability of all patient documentation on the computer screen and through integration of standard report templates with specific details of testing undertaken and its results.