Biomedical subjects

Kashif A Haque

Publications and source records attributed to Kashif A Haque.

5 recordsLinked to original sources

PubMed2006

Long-term storage and recovery of buccal cell DNA from treated cards.

Economical methods for collecting and storing high-quality DNA are needed for large population-based molecular epidemiology studies. Buccal cell DNA collected via saliva and stored on treated filter paper cards could be an attractive method, but modest DNA yields and the potential for reduced recovery of DNA over time were unresolved impediments. Consequently, buccal cell DNA collection via oral mouthwash rinsing became the method of choice in epidemiologic studies. However, the amount of genomic DNA (gDNA) required for genotyping continues to decrease, and reliable whole genome amplification (WGA) methods further reduced the mass of gDNA needed for WGA to 10 ng, diminishing the obstacle of low DNA yields from cards. However, concerns about yield and DNA quality over time remained. We located and analyzed 42 buccal cell saliva samples collected and stored on treated cards for 7 years at room temperature, -20 degrees C, and -80 degrees C. We recovered DNA from the treated cards, estimated the concentration by a human-specific quantitative real-time PCR assay, and evaluated the quality by PCR amplification of 268-, 536-, and 989-bp fragments of the beta-globin gene and by AmpFlSTR Identifiler assay analysis. Most DNA yields per 3-mm punch were <10 ng, and most PCR amplicons failed to amplify, where size of the amplicon was negatively associated with successful amplification. Using these methods, treated cards did not consistently provide sufficient quantities of buccal cell gDNA after 7 years of storage for genotyping or WGA.

DNA↗

PubMed2005

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.

BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.

Biotechnology↗

PubMed2005

Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources.

The promise of whole genome amplification (WGA) is that genomic DNA (gDNA) quantity will not limit molecular genetic analyses. Multiple displacement amplification (MDA) and the OmniPlex PCR-based WGA protocols were evaluated using 4 and 5 ng of input gDNA from 60 gDNA samples from three tissue sources (mouthwash, buffy coat, and lymphoblast). WGA DNA (wgaDNA) yield and genotyping performance were evaluated using genotypes determined from gDNA and wgaDNA using the AmpFlSTR Identifiler assay and N = 49 TaqMan SNP assays. Short tandem repeat (STR) and SNP genotyping completion and concordance rates were significantly reduced with wgaDNA from all WGA methods compared with gDNA. OmniPlex wgaDNA exhibited a greater reduction in genotyping performance than MDA wgaDNA. Reduced wgaDNA genotyping performance was due to allelic (all protocols) and locus (OmniPlex) amplification bias leading to heterozygote and locus dropout, respectively, and %GC sequence content (%GC) was significantly correlated with TaqMan assay performance. Lymphoblast wgaDNA exhibited higher yield (OmniPlex), buffy coat wgaDNA exhibited higher STR genotyping completion (MDA), whereas mouthwash wgaDNA exhibited higher SNP genotyping discordance (MDA). Genotyping of wgaDNA generated from < or = 5 ng gDNA, e.g., from archaeological, forensic, prenatal diagnostic, or pathology samples, may require additional genotyping validation with gDNA and/or more sophisticated analysis of genotypes incorporating observed reductions in genotyping performance.

Adult↗

PubMed2005

Effects of electron-beam irradiation on whole genome amplification.

Electron-beam (E-beam) irradiation, currently being used to sterilize mail addressed to selected ZIP codes in the United States, has significant negative effects on the genomic integrity of DNA extracted from buccal-cell washes. We investigated the yield, composition, and genotyping performance of whole genome amplified DNA (wgaDNA) derived from 24 matched samples of E-beam-irradiated and nonirradiated genomic DNA (gDNA) as a model for the effects of degraded gDNA on the performance of whole genome amplification. gDNA was amplified using the Multiple Displacement Amplification method. Three methods of DNA quantification analysis were used to estimate the yield and composition of wgaDNA, and 65 short tandem repeat and single nucleotide polymorphism genotyping assays were used to evaluate the genotyping performance of irradiated and nonirradiated gDNA and wgaDNA. Compared with wgaDNA derived from nonirradiated gDNA, wgaDNA derived from irradiated gDNA exhibited a significantly reduced yield of wgaDNA and significantly reduced short tandem repeat and single nucleotide polymorphism genotyping completion and concordance rates (P < 0.0001). Increasing the amount of irradiated gDNA input into whole genome amplification improved genotyping performance of wgaDNA but not to the level of wgaDNA derived from nonirradiated gDNA. Multiple Displacement Amplification wgaDNA derived from E-beam-irradiated gDNA is not suitable for genotyping analysis.

DNA↗

PubMed2003

Performance of high-throughput DNA quantification methods.

BACKGROUND: The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of approximately 350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. RESULTS: The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0-95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8-17.5%). Residual error (3.2-59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures. CONCLUSION: The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

Analysis of Variance↗