Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories

Abstract

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities, their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic DNA Collection in order to address specific health priorities through molecular techniques. Our approach employs a low-cost laundry detergent based DNA extraction technique that maximizes DNA yield and quality. We have optimised four different modalities (maxiprep, midiprep, miniprep and microprep) for two different sources (leukocyte concentrates and whole blood). Our optimised protocol produces 4.5 mg of DNA from 15 ml of blood-bank discarded leukocyte concentrates with spectrophotometric quality, genomic integrity and PCR suitability that rivals that of phenol-chloroform extracted samples. We present evidence of many PCR applications that we have carried out on samples extracted with this technique including Killer-cell Immunoglobulin-like Receptor genotyping, Short Tandem Repeat profiling as well as nucleic acid screening for hepatitis B and human immunodeficiency type-1 viruses. This paper highlights many of the advantages that this DNA extraction technique provides over existing methodologies, whether it is used to establish large genomic DNA collections (as was our main intention) or as a routine DNA extraction method for PCR applications.

KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation

“…Relative GAPDH levels were then used to generate a normalization factor for each extracted DNA sample relative to the average for the experiment. Normalized KSHV genomes per 35 ng of total extracted DNA were then further converted to relative copies per 1,000 cells by an estimate of 9 pg of genomic DNA per cell (81)(82)(83). Standard deviation of the absolute viral genome copy number across triplicate wells was propagated through these normalization calculations.…”

Evaluation of tacrolimus‐related CYP3A5 genotyping in China: Results from the First External Quality Assessment Exercise

“…A previously described method was used to culture the immortalized cell lines . An optimized DNA extraction technique that maximizes DNA yield and quality was used to isolate genomic DNA from the cells . Tris‐EDTA buffer was used for the resuspension of the extracted genomic DNA.…”

Association of KIR3DL1/S1 and HLA-Bw4 with CD4 T cell counts in HIV-infected Mexican mestizos

“…KIR and HLA genotyping Genomic DNA was extracted from EDTA anticoagulated peripheral blood as described previously (Garcia-Sepulveda et al 2010). KIR, HLA-B public epitope typing and broad HLA-C group 1 or 2 typing were performed using previously published SSP-PCR-based strategies (Alvarado-Hernandez et al 2011;Bunce et al 1995;Hiby et al 2008).…”