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Principal Investigator
Name
Leah Mechanic
Institution
Westat
Email
About this CDAS Project
Study
PLCO (Learn more about this study)
Project ID
2008-0042
Initial CDAS Request Approval
Oct 9, 2008
Title
DNA Extraction Method, Biological Specimen, and DNA Yield for Genetic Studies in PLCO
Summary
The field of genetic epidemiology has witnessed a revolution due to the launch of the International HapMap project describing the linkage disequilibrium of the human genome in 2002 and the advent of less expensive, high density genotyping platforms. These platforms enable genotyping of millions of single nucleotide polymorphisms (SNPs) in a single individual. The quality of DNA used in genotyping assays influences genotyping success and may influence the results. We hypothesize that the usability of DNA for genomic assays, a surrogate for DNA quality, was modulated by the type of biological specimen, method of DNA extraction, and variation in collection and storage. We will investigate this hypothesis using data obtained from specimens selected for a case-control study nested within the Prostate Lung Colorectal and Ovarian (PLCO) screening trial examining association of genetic variation with breast cancer. DNA quantity was assessed using Nanodrop or PicoGreen assay results. Specimens with DNA concentrations greater than 25 ng/ml and total yields of at least 20 mg were considered suitable (or usable) for genotyping based on standards established by the NCI Core Genotyping Facility (http://cgf.nci.nih.gov/). The number of suitable DNA samples for genotyping purposes and average DNA amounts per volume of starting specimen obtained after DNA extraction will be compared for each method of DNA extraction and type of biological specimen. The association of DNA usability with time between blood collection and freezing and storage time will also examined. Determining how DNA quantity and usability are affected by preanalytic variables, such as extraction method, type of biological specimen, time between blood collection and freezing, and storage time will enable investigators to optimize the design and implementation of genetic association and candidate gene studies.
Aims

High yields of DNA are required for high throughput genotyping assays, such as whole genome scans and more sensitive genetic assays, including measuring genetic methylation or somatic mutations. We hypothesize that DNA usability for genomic assays is modulated by the type of biological specimen (whole blood, buffy coat, buffycoat/RBC), method of DNA extraction, and variation in collection and storage. For the purposes of this proposal, DNA usability, or suitable for genotyping, is defined according to standards used by the NCI Core Genotyping Facility (CGF) (http://cgf.nci.nih.gov/) as specimens with concentrations greater than 25 ng/ml and total yields of at least 20 mg. This hypothesis will be addressed by the following aims: Aim 1. Examine the yields of DNA obtained using Qiagen, Gentra Autopure or Phenol-Chloroform extraction obtained from buffy coat specimens. The usability of DNA will be evaluated by examining the average amount of DNA (total amount, concentration) obtained from one vial of buffy coat specimens as estimated using the Nanodrop assay. Aim II. Determine the relationship between the biological specimen and DNA usability. DNA yields and quality, evaluated as described in Aim I, will be compared for whole blood, buffy coat, red blood cell (RBC)/buffy coat specimens obtained in PLCO using the Qiagen method for DNA purification. Aim III. Investigate how increasing the amount of time post-collection before a whole blood specimen is processed and/or the length of time a fractionated whole blood sample is in storage before its DNA is extracted affects the usability of the resulting DNA. The storage time of whole blood specimens and the time between blood draw and freezing of the specimen will be compared with DNA yield. Another potential source of variation to be examined is different screening center where specimens were collected. Some screening centers processed blood in satellite centers or while others process blood at major hospital labs or SC site-specific labs.

Collaborators

Arti Varanasi (Westat)
Asia Khan (Westat)
Barbara O'Brien (Westat)
Danielle Carrick (Westat)
Karen Pettit (Westat)
Leah Mechanic (Westat)
Mark Cosentino (SAIC-Frederick)
Tim Sheehy (SAIC-Frederick)