Project leader: Dr Helen White

NHS genetic diagnostic laboratories perform thousands of tests every month using a variety of technologies. Laboratories generally utilise locally developed controls as standards to confirm that the assay is working correctly. Consequently there is a degree of variation in the number and type of controls employed in different laboratories which could potentially compromise quality assurance. To address this problem NGRL Wessex are generating sets of plasmid constructs that harbour defined sequence changes and which can be used as controls for a wide range of mutation assays. A set of pilot constructs for analysis of hMLH1 and BRCA2 were sent to 13 interested UKGTN laboratories in May 2003 for performance evaluation.

Provided that these reagents are found to be of value, we envisage that they will be put through further rounds of development and evaluation prior to establishment as defined control reagents. Ultimately we aim to establish these controls as certified reference materials (CRMs) in conjunction with CRMGEN (an EU-funded consortium lead by Dr David Barton, Dublin), but this depends on the resolution of a number of issues, notably potential restrictions/costs that may be imposed by the holders of gene patents. Possible routes to the establishment of CRMs via NIBSC (South Mimms) and IRRM (Geel, Belgium) are under investigation.

Generic Mutation Detection Reference Reagents ^New

To facilitate the evaluation of high throughput mutation detection strategies that are currently being introduced into UKGTN labs we have developed a generic set of reference reagents which can be used to assess both new and existing mutation detection techniques. Plasmid controls have been produced which can be used to determine the sensitivity and specificity of these techniques by analysing factors that are of general importance for all technologies including: the type of base substitution, the GC content of the amplicon and the location of the mutation in the fragment. We are currently using these reagents to evaluate CSCE and dHPLC and will be running a more extensive field trial early in 2005.

The controls can be used to amplify fragments ranging from 400-450bp with an average GC content of 20%, 40%, 60% and 80%. The wild type sequence has been mutated to produce every possible heteroduplex (8 in total) at three positions within the amplicon as shown in figure 1.

Production of Plasmid Constructs:

Blood taken from 8 lab volunteers

DNA extracted and pooled

Normal DNA sequences amplified using PCR

PCR products cloned into T-tailed vector

Verification of normal sequence

Mutations introduced using site directed mutagenesis

Verification of mutant sequence

Evaluation of constructs by dHPLC

Plasmid Constructs available from Wessex NRGL:

The following control plasmid mixes are currently available to UKGTN laboratories on request. Enquiries from other centres or commercial organisations should be made to Dr Helen White. For details of the cloned fragments and examples of dHPLC profiles of the mutated exons click on the hyperlinks below:

The controls are supplied as wild type and mutant plasmid mixes with each plasmid diluted to 10⁴ copies/µl in 0.1XTE containing 50µg/ml tRNA as a carrier. This dilution is equivalent to the copy number found in 100ng genomic DNA and therefore the plasmids should not pose a contamination threat greater than patient DNA samples.

Protocol for using plasmid controls

Downloads:
BRCA2 mRNA sequence with position of all mutations annotated
hMLH1 mRNA sequence with position of all mutations annotated
BRCA1 mRNA sequence with position of all mutations annotated
hMSH2 mRNA sequence with position of all mutations annotated