New technique for rapid mutation detection

23 December 2008

The gold standard for mutation screening for many years has been amplification of target sequences by polymerase chain reaction (PCR) followed by Sanger sequencing. However, when it comes to conditions where the mutation may be in one of several genes or polygenic conditions where a number of different genes may be involved (e.g. breast cancer), gathering results by this technique can often be labour intensive and time consuming as numerous different genes have to be sequenced. Although a number of parallel sequencing techniques have been developed, along with “front-end” methods to amplify a subset of genes (see previous news), these procedures are still too expensive and complex to be applied in a routine diagnostic or screening setting.

Recently, VIB researchers at the University of Antwerp have developed a new method of detecting mutations and copy number variations that may make diagnosis of inherited diseases quicker (see press release). The technique involves a combination of multiplex PCR (amplification of multiple targets by using more than one set of primers) with high-throughput parallel sequencing, in order to analyse multiple genes simultaneously. In a paper published this month, Goossens et al. describe the use of this technique to identify mutations in seven genes associated with Charcot-Marie Tooth Disease (CMT) – an inherited disorder that affects the peripheral nervous system [Goossens et al 2008 Hum Mutat. 0(1-6)]. The researchers amplified and sequenced seven genes from 31 individuals and compared the mutations detected with the results obtained by traditional PCR-based Sanger sequencing. All the variants that were detected by traditional methods were also identified using the new technique. They were also able to determine the copy number variation (CNV) of two genes associated with CMT (GJB1 and PMP22).

Comment: Although this new method still needs validating, if successful it could make diagnosis and screening for many inherited conditions much more rapid (weeks as opposed to months). In addition, with inherited conditions that can be a result of mutations in several genes, where mutation screening is usually only undertaken to identify common variants, this technique may allow screening for a larger number of variants at a fraction of the cost.

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