Use of robotics in high-throughput DNA sequencing.

Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation pre-screening step to limit the amount of DNA sequencing required. Protocols to pre-screen for mutations and limit the amount of DNA sequencing may not localise every base change present and/or require considerable levels of manual intervention.

Long-PCR amplification of human genomic DNA.

Standard polymerase chain reaction (PCR) protocols amplify relatively small fragments precluding the use of this approach when examining gross rearrangements of DNA. By using combinations of DNA polymerases, which feature either good polymerase activity or error-correction abilities, it is now possible to extend the length of DNA fragment that can be amplified. These "long-PCR" protocols have allowed the development of more rapid and convenient ways to analyse large-scale rearrangements of DNA and in many cases has superseded alternative approaches such as Southern blotting.

A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3.

Direct sequencing of VWF genomic DNA in 21 patients with type 3 von Willebrand disease (VWD) failed to reveal a causative homozygous or compound heterozygous VWF genotype in 5 cases. Subsequent analysis of VWF mRNA led to the discovery of a deletion (c.221-977_532 + 7059del [p.

An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease.

Forty families diagnosed by UK centres to have type 1 VWD were recruited. Following review, six families were re-diagnosed to have type 2 VWD, one to have a platelet storage pool disorder, and one family was determined to be unaffected. Direct DNA sequencing of the promoter region and all exons and intronic boundaries of the VWF gene identified six mutations likely to be causative of VWD in index cases of nine of the 32 (28%) confirmed type 1 VWD families.