Isolation and characterization of DNA from the plasma of cancer patients

doi:10.1016/0277-5379(87)90266-5

European Journal of Cancer and Clinical Oncology

Volume 23, Issue 6, June 1987, Pages 707-712

European Journal of ...

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Abstract

Ten out of 37 patients with advanced malignant diseases were found to have extractable amounts of DNA in their plasma whereas no DNA could be detected in 50 normal controls. After its purification from the original nucleoprotein complex, DNA plasma levels ranging from 0.15 to 12 μg/ml were measured, the lowest concentration detectable with our method being 0.1 μg/ml. Knowing from recovery experiments performed with ³²P-DNA that the loss of DNA during the extraction procedure is about 65%, the real concentration of DNA in the plasma corresponds to about 3 times the given figures. The purified DNA was shown to be double-stranded and composed of fractions ranging from 21 kb to less than 0.5 kb, as determined by agarose gel electrophoresis. All these fractions hybridized with a ³²P-labelled human DNA probe indicating the human origin of the bulk of the circulating DNA. In conclusion: the finding of extractable amounts of DNA in the plasma of 27% of the investigated cancer patients, and its absence from the controls, suggests some correlation with malignancy.

References (18)

AR Neurath et al.
Strategies for detection of transfusion-transmitted viruses eluding identification by conventional serologic tests. II. Detection of host DNA in human plasmas with elevated alanine aminotransferase
J Virol Methods
(1984)
M Edelman
Purification of DNA by affinity chromatography: removal of polysaccharide contaminants
Analyt Biochem
(1975)
M Stroun et al.
Circulating nucleic acids in higher organisms
Int Rev Cytol
(1977)
CR Steinman
Circulating DNA in systemic lupus erythematosus
J Clin Invest
(1984)
SA Leon et al.
Free DNA in the serum of cancer patients and the effect of therapy
Cancer Res
(1977)
B Shapiro et al.
Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease
Cancer
(1983)
NA Carpentier et al.
The presence of circulating DNA in patients with acute or chronic leukemia: relation to serum anti-DNA antibodies and Cl_q binding activity
Human Lymphocyte Differentiation
(1981)
P Anker et al.
Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system
Cancer Res
(1975)
DWJ Rigby et al.
Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I
J Mol Biol
(1977)

There are more references available in the full text version of this article.

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Citation Excerpt :
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European Journal of Cancer and Clinical Oncology

Abstract

References (18)

Strategies for detection of transfusion-transmitted viruses eluding identification by conventional serologic tests. II. Detection of host DNA in human plasmas with elevated alanine aminotransferase

J Virol Methods

Purification of DNA by affinity chromatography: removal of polysaccharide contaminants

Analyt Biochem

Circulating nucleic acids in higher organisms

Int Rev Cytol

Circulating DNA in systemic lupus erythematosus

J Clin Invest

Free DNA in the serum of cancer patients and the effect of therapy

Cancer Res

Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease

Cancer

The presence of circulating DNA in patients with acute or chronic leukemia: relation to serum anti-DNA antibodies and Cl_q binding activity

Human Lymphocyte Differentiation

Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system

Cancer Res

Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

J Mol Biol

Cited by (306)

New insights into nucleic acid sensor AIM2: The potential benefit in targeted therapy for cancer

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis

Blood has never been thicker: Cell-free DNA fragmentomics in the liquid biopsy toolbox of B-cell lymphomas

Circulating DNA fragmentomics and cancer screening

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers

When Tissue Is the Issue: Expanding Cell-Free DNA “Liquid Biopsies” to Supernatants and Nonplasma Biofluids

Isolation and characterization of DNA from the plasma of cancer patients

Abstract

J Virol Methods

Analyt Biochem

Int Rev Cytol

Circulating DNA in systemic lupus erythematosus

J Clin Invest

Free DNA in the serum of cancer patients and the effect of therapy

Cancer Res

Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease

Cancer

The presence of circulating DNA in patients with acute or chronic leukemia: relation to serum anti-DNA antibodies and Clq binding activity

Human Lymphocyte Differentiation

Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system

Cancer Res

Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

J Mol Biol

The presence of circulating DNA in patients with acute or chronic leukemia: relation to serum anti-DNA antibodies and Cl_q binding activity