Selective isolation of mercurated DNA by affinity chromatography on thiol matrices

G. Bánfalvi, Sudha Bhattacharya, Nilima Sarkar

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

A method for isolating picomole quantities of nascent mercurated DNA from a mixture of cellular nucleic acids using affinity chromatography on thiol-agarose is described. Analysis of mercurated DNA (HgDNA) isolated in the presence of in vivo-labeled cellular RNA or in vitro-synthesized RNA showed a low level of RNA contamination, about 0.04-0.16%, in the HgDNA. Comparative binding studies on different thiol matrices showed that the efficiency of binding of HgDNA was related to the nature but not to the SH content of the matrix used. Another important parameter for binding was the structure of HgDNA. The recovery was 98% with large nascent HgDNA sedimenting at about 30 S, whereas for short pulse-labeled single-stranded HgDNA (20-50 nucleotides long), the maximum recovery was 60%. The effect of the structure of HgDNA on the binding to the thiol matrix was probed using a variety of well-defined mercurated structures obtained from phage DNA and their restriction fragments. For DNA containing one 5-mercuricytidine 5′-triphosphate (HgdCMP) residue at each 3′-end, short fragments (size range, 230-510 bp) were bound quantitatively. With larger fragments (size range, 490-1100 bp), the binding decreased progressively with increasing size. DNA fragments larger than 1060 bp did not bind to the matrix. Single-stranded DNA containing only one HgdCMP at one end did not bind to the matrix even in the size range 200-1100 nucleotides. In contrast, continuous stretches of HgdCMP residues in one strand or short stretches of HgdCMP residues at random in both strands permit quantitative binding irrespective of size. Mercuration of DNA has been achieved by three procedures: in vitro replication, nick translation, and end labeling with HgdCMP of the recessed 3′-ends of restriction fragments. Thus, highly purified HgDNA can be isolated by the procedures described which are simple, efficient, and reproducible.

Original languageEnglish
Pages (from-to)64-70
Number of pages7
JournalAnalytical Biochemistry
Volume146
Issue number1
DOIs
Publication statusPublished - 1985

Fingerprint

Affinity chromatography
Affinity Chromatography
Sulfhydryl Compounds
DNA
RNA
Nucleotides
Recovery
Bacteriophages
Single-Stranded DNA
Sepharose
Labeling
Nucleic Acids
Contamination

Keywords

  • affinity chromatography
  • affinity labeling
  • DNA
  • DNA polymerase
  • nucleic acid chemistry
  • purines and pyrimidines

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Selective isolation of mercurated DNA by affinity chromatography on thiol matrices. / Bánfalvi, G.; Bhattacharya, Sudha; Sarkar, Nilima.

In: Analytical Biochemistry, Vol. 146, No. 1, 1985, p. 64-70.

Research output: Contribution to journalArticle

Bánfalvi, G. ; Bhattacharya, Sudha ; Sarkar, Nilima. / Selective isolation of mercurated DNA by affinity chromatography on thiol matrices. In: Analytical Biochemistry. 1985 ; Vol. 146, No. 1. pp. 64-70.
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abstract = "A method for isolating picomole quantities of nascent mercurated DNA from a mixture of cellular nucleic acids using affinity chromatography on thiol-agarose is described. Analysis of mercurated DNA (HgDNA) isolated in the presence of in vivo-labeled cellular RNA or in vitro-synthesized RNA showed a low level of RNA contamination, about 0.04-0.16{\%}, in the HgDNA. Comparative binding studies on different thiol matrices showed that the efficiency of binding of HgDNA was related to the nature but not to the SH content of the matrix used. Another important parameter for binding was the structure of HgDNA. The recovery was 98{\%} with large nascent HgDNA sedimenting at about 30 S, whereas for short pulse-labeled single-stranded HgDNA (20-50 nucleotides long), the maximum recovery was 60{\%}. The effect of the structure of HgDNA on the binding to the thiol matrix was probed using a variety of well-defined mercurated structures obtained from phage DNA and their restriction fragments. For DNA containing one 5-mercuricytidine 5′-triphosphate (HgdCMP) residue at each 3′-end, short fragments (size range, 230-510 bp) were bound quantitatively. With larger fragments (size range, 490-1100 bp), the binding decreased progressively with increasing size. DNA fragments larger than 1060 bp did not bind to the matrix. Single-stranded DNA containing only one HgdCMP at one end did not bind to the matrix even in the size range 200-1100 nucleotides. In contrast, continuous stretches of HgdCMP residues in one strand or short stretches of HgdCMP residues at random in both strands permit quantitative binding irrespective of size. Mercuration of DNA has been achieved by three procedures: in vitro replication, nick translation, and end labeling with HgdCMP of the recessed 3′-ends of restriction fragments. Thus, highly purified HgDNA can be isolated by the procedures described which are simple, efficient, and reproducible.",
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