A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 72 ·No. 1 ·1975-01-00 ·Pages 83-7

Vollenweider HJ, Sogo JM, Koller T

Abstract

A protein-free nucleic acid preparation method for electron microscopy is described. The basic procedure is very similar to the classical protein monolayer spreading techniques. The carrier protein (usually cytochrome c) is replaced by benzyldimethylalkylammonium chloride. Both the hypophase method and the microdiffusion or droplet method can be applied with this compound. Unlike cytochrome c, benzyldimethylalkylammonium chloride does not lead to any apparent thickening of the nucleic acid strands. Partially denatured DNA spread with this reagent shows a loosened structure with a foamy appearance in the regions previously considered to be "unmelted," which open up locally into melted loops of different size. Specifically bound proteins, such as RNA polymerase on bacteriophage T7 DNA, can be detected unambiguously.

MeSH Terms

Coliphages Cytochrome c Group Cytological Techniques DNA Viruses DNA, Single-Stranded DNA, Viral Microscopy, Electron/methods Nucleic Acid Denaturation Nucleic Acids Nucleoproteins Protein Binding

Chemicals

Cytochrome c Group DNA, Single-Stranded DNA, Viral Nucleic Acids Nucleoproteins

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Vollenweider H J

Sogo J M

Koller T

References (13)

13 references, click to expand

Determination of base sequence in nucleic acids with the electron microscope: visibility of a marker.
Proc Natl Acad Sci U S A. 1962 Mar 15;48:409-16 PMID: 13866528
Electron microscopy of size and shape of viral DNA in solutions of different ionic strengths.
J Mol Biol. 1967 Jan 28;23(2):163-81 PMID: 4292655
Molecular weights of coliphages and coliphage DNA. 3. Contour length and molecular weight of DNA from bacteriophages T4, T5 and T7, and from bovine papilloma virus.
J Mol Biol. 1970 Dec 28;54(3):557-65 PMID: 4321637
Electron-microscopic localization of the binding of Escherichia coli RNA polymease to T7 DNA in vitro.
J Mol Biol. 1972 Aug 21;69(2):179-86 PMID: 4560945
Electronmicroscopy of genetic activity.
Annu Rev Biochem. 1973;42:379-96 PMID: 4581229
Mechanism of viral replication. I. Structure of replication complexes of R17 bacteriophage.
J Mol Biol. 1973 Dec 15;81(3):367-80 PMID: 4588279
Electron microscopy of single-stranded DNA.
J Mol Biol. 1970 Apr 14;49(1):125-37 PMID: 5450511
An electron microscopic method for studying nucleic acid-protein complexes. Visualization of RNA polymerase bound to the DNA of bacteriophages T7 and T3.
Biopolymers. 1974 May;13(5):995-1009 PMID: 4604084
Binding sites of E. coli RNA polymerase on T7 DNA as determined by electron microscopy.
FEBS Lett. 1974 Sep 1;45(1):64-7 PMID: 4606271
A new preparation method for dark-field electron microscopy of biomacromolecules.
J Ultrastruct Res. 1971 Apr;35(1):147-67 PMID: 4931423
The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT).
Biopolymers. 1972;11(4):745-59 PMID: 5028511
Simplified quantitative electron microscopy of biopolymers.
Biopolymers. 1970;9(3):373-9 PMID: 5417647
Electron microscopic localization of the binding sites of Escherichia coli RNA polymerase in the early promoter region of T7 DNA.
Eur J Biochem. 1974 May 15;44(2):617-24 PMID: 4600365

Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.72.1.83

Published

1975-01-00

Pages

83-7

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC432245

Subset

External Links

PubMed source DOI: 10.1073/pnas.72.1.83 PMC Full Text View journal details

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A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules.

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