Effect of HIV-1 infection on human DNA yield from saliva.

PURPOSE: Saliva is a good source of DNA for genomic research, and leukocytes are a predominant source of DNA in human saliva. Advanced human immunodeficiency virus (HIV)-type 1 infection disrupts tonsillar architecture and depletes tonsillar lymphocytes. We tested whether HIV-1 infection reduces extracted human DNA yield from saliva. METHODS: Approximately 2 mL of expectorated saliva was collected from HIV-infected adults during routine primary care clinic visits and from healthy, HIV-negative controls. Human DNA was manually extracted and was specifically quantified by assaying for the RNAse P gene. RESULTS: Seventy-five individuals were studied, including 25 HIV-infected adults with <200 CD4+ T cells/mm(3) (i.e., acquired immunodeficiency syndrome), 25 with >200 CD4+ T cells/mm3, and 25 HIV-negative controls. Overall DNA yield was 64.7 microg [29.0-139.7 microg] (median [interquartile range]). Yields were comparable among HIV-infected individuals with lower CD4+ T cell counts (74.3 microg [39.4-151.4 microg]), higher CD4+ T cell counts (63.9 microg [29.2-172.1 microg]), and HIV-negative controls (61.4 microg [28.4-123.4 microg]) (p > .05). CONCLUSION: Infection with HIV-1 does not reduce human DNA yield from saliva. Expectorated saliva should provide sufficient extracted native DNA for genomic studies in HIV-infected individuals.

Whole-genome amplification of oral rinse self-collected DNA in a population-based case-control study of breast cancer.

The availability of large amounts of genomic DNA (gDNA) is the limiting factor for many of the molecular biology assays in genetic epidemiologic studies. Whole-genome amplification using multiple displacement amplification is used to amplify a representative sample of gDNA from small amounts of gDNA to optimize gDNA yield. We collected oral rinse DNA samples through the mail from 3,377 women enrolled in a population-based U.S. breast cancer case-control study and did whole-genome amplification by multiple displacement amplification. Genotyping was done for 66 single nucleotide polymorphisms (SNP) in 18 candidate susceptibility genes using amplified DNA with genomic replicates included for quality control. The concordance rates (percentages of agreement) in 95 quality control replicates of gDNA and amplified DNA for 66 SNPs ranged from 88% to 100% (median, 97%). The average allelic error rate was 0.9%. However, in further analyses based on the full control series (n = 1,492), >60% of the SNPs failed tests for Hardy-Weinberg equilibrium (P < 0.05), with evidence of heterozygote loss in the great majority. Even eliminating the 9% of samples with lower quality or input DNA, tests for Hardy-Weinberg equilibrium indicated persistent allele bias in nearly a third of the SNPs. Whole-genome amplification may introduce substantial allele amplification bias in gDNA collected using a common protocol in population-based epidemiologic studies.

Phenotype-Driven Plasma Biobanking Strategies and Methods.

Biobank development and integration with clinical data from electronic medical record (EMR) databases have enabled recent strides in genomic research and personalized medicine. BioVU, Vanderbilt's DNA biorepository linked to de-identified clinical EMRs, has proven fruitful in its capacity to extensively appeal to numerous areas of biomedical and clinical research, supporting the discovery of genotype-phenotype interactions. Expanding on experiences in BioVU creation and development, we have recently embarked on a parallel effort to collect plasma in addition to DNA from blood specimens leftover after routine clinical testing at Vanderbilt. This initiative offers expanded utility of BioVU by combining proteomic and metabolomic approaches with genomics and/or clinical outcomes, widening the breadth for potential research and subsequent future impact on clinical care. Here, we describe the considerations and components involved in implementing a plasma biobank program from a feasibility assessment through pilot sample collection.