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J Mol Diagn. 2006 Feb;8(1):22-30.

Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.

Author information

1
Department of Radiation Oncology, Dana-Farber/Brigham and Women's Cancer Center, Brigham and Women's Hospital, Level L2, Radiation Therapy, 75 Francis St., Boston, MA 02115, USA.

Abstract

Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by approximately 1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens.

PMID:
16436631
PMCID:
PMC1867563
DOI:
10.2353/jmoldx.2006.050074
[Indexed for MEDLINE]
Free PMC Article

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