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A Method for Characterization of Total Circulating DNA

Authors


Address for correspondence: Piet J. Pretorius, Private Bag ×6001 Potchefstroom 2520 South Africa. Voice: +27(0)18-299-2309; fax: +27(0)18-299-2316. piet.pretorius@nwu.ac.za

Abstract

Although much work has been done in the field of circulating DNA, no definitive information on sequencing data of total circulating DNA is available. Characterization of total circulating DNA by sequence analysis may give valuable information about the origin and function of these nucleic acid molecules. Circulating DNA was isolated from plasma of one healthy individual and one cancer patient with various methods and was cloned into a blunted cloning vector. Resulting colonies were sequenced and analyzed. The majority of the DNA that ligated into the vector was about 200 bp in length. Sequence analysis revealed that circulating DNA consists partly of currently uncharacterized human genomic sequences and when human repeats were masked it matched with sequences present in contigs containing known genes situated at various distances from the identified targets. In addition to the presence of large repeats, a variety of Alu repeat sequences were observed. Preliminary results showed that more Alu repeats are present in the plasma of normal individuals than in patient material. None of the gene sequences reported in the literature to be part of circulating DNA (e.g., P53, the Ras family, β-globin, or β-actin) was observed. Cloning and sequencing of free circulating DNA was successful and this first attempt on characterizing sequence data of free circulating DNA not only confirmed previous results, but also revealed a large variety of sequences. Further characterization of circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA.

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