Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-09-02
1996-04-30
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435180, C12Q 168, G01N 118
Patent
active
055124364
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP92/01479, filed on Jun. 30, 1992, which is claiming priority to GB 91144180.4, filed on Jul 1, 1991.
The present invention concerns the hybridization between two complementary polynucleotide segments, and relates in particular to agents for increasing the rate of hybridization and total amount of hybrid formed.
The use of dextran sulphate to increase the rate of DNA hybridization has been reported by a number of workers. U.S. Pat. No. 4,302,204 describes the use of dextran sulphate and other charged polysaccharides to accelerate hybridization reactions in which one of the polynucleotides is covalently attached to a solid phase.
Polyethylene glycol was found by Renz and Kurz (1984) Nucleic Acid Research 12, 3435-3444, to be superior to dextran sulphate for hybridizations with peroxidase labelled nucleic acids. However, subsequent experiments by the author of the present invention where the conditions and concentration of the other components in the hybridization buffer have been altered, showed that dextran sulphate was superior to polyethylene glycol when they were used as the sole hybridization rate enhancers.
U.S. Pat. No. 4,689,294 discloses the use of polyacrylate and polymethacrylate in hybridization buffers, and it states that these two polymers have the following properties:
a) the same rate of hybridization enhancement as dextran sulphate,
b) resistance to microbial degradation,
c) non-specific binding of probe, to the commonly used nitrocellulose supports, is substantially lower, compared to use of dextran sulphate,
d) Polyacrylate is effective at low concentrations and is significantly less expensive than dextran sulphate.
An experiment was performed by the author of the present invention in which it was attempted to hybridize a peroxidase-labelled nucleic acid probe, from solution, to a nucleic acid bound to a charged nylon solid support. It was found that with polyacrylate in the hybridization buffer at 5% concentration, the hybridization of the probe of the complementary nucleic acid bound to the solid support was prevented. Thus, polyacrylates are unsuitable at least for membrane hybridization using peroxidase-labelled probes, and Southern blots made with charged nylon solid support.
The inventor therefore undertook a study to determine if other rate enhancers could be found which would perform as well as dextran sulphate when used at concentrations of 5% (w/v) or less in the hybridization buffer.
BRIEF SUMMARY OF THE INVENTION
It has now been found that polyvinyl alcohol and polystyrene sulphonic acid also increase the rate and/or extent of hybridization of complementary polynucleotide segments. Accordingly in one aspect the present invention provides a method of hybridizing complementary polynucleotides which method comprises maintaining the complementary polynucleotides in a buffered aqueous medium under hybridization conditions, characterized in that the buffered aqueous medium contains polyvinyl alcohol and/or polystyrene sulphonic acid at a concentration to produce an observable increase in the rate and/or extent of hybridization.
In another aspect, the invention provides a hybridization buffer characterized by containing polyvinyl alcohol and/or polystyrene sulphonic acid at a concentration effective to produce an observable increase in the rate and/or extent of hybridization of complementary polynucleotides.
DETAILED DESCRIPTION
The two polymers, polyvinyl alcohol and polystyrene sulphonic acid, may be present in the buffered aqueous medium in anionic form which can be formed in situ or by using a salt of the polymers e.g. Na, K or NH.sub.4 salts. A preferred salt is the Na salt. The term polystyrene sulphonic acid is thus used herein to include polystyrene sulphonate.
The hybridization buffer may be of conventional composition, and may also contain various other components which are conventionally used in hybridization media such as surfactants and other polymers.
The polyvinyl alcohol and polystyrene sulphonic acid will normally be present in
REFERENCES:
patent: 4302204 (1981-11-01), Wahl et al.
patent: 4689294 (1987-08-01), Boguslawski et al.
Zhov et al, Appl. Environ. Microbiol 57:2963-2968 (1991).
Maniatis et al "Molecular Cloning" (1982) pp. 326-327.
JP 2119800, May 7, 1990, "Method of Detection Nucleic Acid . . . " (Derwent Biotech Abs).
Nucleic Acids Research, vol. 14, No. 18, 1986, pp. 7285-7303; N. Casna et al: "Genomic Analysis II: Isolation of High Molecular Weight Heteroduplex DNA Following Differential Methylase Protection and Formamide-Pert Hybridization".
Amersham International plc
Campbell Eggerton
Jones W. Gary
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