Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry.

Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from volunteer buccal swabs but the system has not been tested using more challenging sample types. For mass spectrometry to be considered as a valid alternative to Sanger sequencing it must also be demonstrated to be suitable for use with more limiting sample types such as old teeth, bone fragments, and hair shafts. In 2009 the Commonwealth War Graves Commission launched a project to identify the remains of 250 World War I soldiers discovered in a mass grave in Fromelles, France. This study characterises the performance of both Sanger sequencing and the T5000 platform for the analysis of the mitochondrial DNA extracted from 225 of these remains, both in terms of the ability to amplify and characterise DNA regions of interest and the relative information content and ease-of-use associated with each method.

Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes.

BACKGROUND: Coumarin anticoagulants such as warfarin are used to treat and prevent thromboembolic events in patients. The required dosage is difficult to predict and the risk of over or under anticoagulation are dependent on several environmental and clinical factors, such as concurrent medication, diet, age and genotype for polymorphisms in two genes CYP2C9 and VKORC1. METHODS: A novel fluorescent PCR genotyping assay using HyBeacon® probes, was developed to enable clinical staff to genotype the CYP2C9*2 and CYP2C9*3 alleles and the VKORC1 G-1639A polymorphism directly from unextracted blood samples. A prototype PCR instrument, Genie 1, suitable for point of care use was developed to carry out the assays. The panel of tests was validated by analysing blood samples from 156 individuals and comparing genotypes with data obtained using DNA samples from the same individuals. The accuracy of genotypes obtained with the Genie 1 was compared against results from well validated real time PCR and PCR-restriction fragment length polymorphism analysis. RESULTS: Identical results were obtained for the newly developed HyBeacon® method and the validation method in all cases except for one where no result was obtained for the VKORC1 polymorphism on the Genie instrument. The samples used for validation represented all six possible *2 and *3 allele-related CYP2C9 genotypes and all three VKORC1 G-1639A genotypes. CONCLUSIONS: We observed excellent accuracy for the newly developed method which can determine genotype in less than 2 h.