Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-07-21
2000-10-03
Yucel, Remy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C12Q 168
Patent
active
061271225
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for high-sensitive silver staining, more specifically, to an improved method for silver staining of DNA which comprises the steps of linking DNA to a polymer which has a high affinity for the DNA and binding of a plenty of silver ions to the DNA-polymer complex.
2. Description of the Prior Art
In general, silver staining is realized by carrying out selective reducing silver ions which are linked to a target material of interest by employing chemical or light (see: Beidler, J. L. et al., Anal. Biochem., 126:374(1982); Goldman. D. and Merril, C. R., Electrophoresis, 3:24(1982)) and detecting light-scattered color of the silver grains produced therefrom (see: Merril, C. R. et al., Proc. Natl. Acad. Sci., USA, 85:453(1988)).
Silver staining methods currently used are largely classified as the following: an alkaline method which reduces a diamine complex of silver nitrate formed under a strong alkaline condition by subjecting the complex in a weakly acidic solution containing formaldehyde; and, an acidic method which reduces silver nitrate in a formaldehyde solution (see: Bassam, B. J. et al., Applied Biochemistry and, Biotechnology, 42:181(1993)).
Although the silver staining technique, at the begining, was developed to detect traces of protein electrophoresed on acrylamide gel (see: Switzer, R. C. et al., Anal. Biochem., 98:231(1979)), through continuous improvement and modification, it has been universally used for detection of lipopolysaccharides (see: Tsai, C. and Frasch, C. E., Anal. Biochem., 119:115(1982)) and nucleic acids (see: Somerville, L. L. and Wang, K., Biochem. Biophys. Res. Commun., 102:54(1981), and proteins as well.
Until now, a variety of silver staining methods for the detection of DNA have been developed as the following: a diamine complex silver staining method (see: Wary, W. et al., Anal. Biochem., 118:197(1981)); an acidic silver staining method (see: Heukeshoven, J. and Dernick, F., Electrophoresis, 6:103(1985); Gottlieb, M. and Chavco, K., Anal. Biochem., 165:33(1987); Bassam, B. J. et al., Anal. Biochem., 98:231(1979)); a cupric-silver method (see: Switzer, R. C. et al., Anal. Biochem., 98:231(1979)) and the like. These silver staining methods are, however, proven to be less satisfactory in the sense that they cannot detect DNA to the level of ng and are not reliable in a view of accuracy and reproducibility.
Accordingly, there are strong reasons for exploring and developing an improved method for detecting DNA with a high sensitivity.
SUMMARY OF THE INVENTION
In this regard, the present inventors, based on Bassam et al's method for silver staining of DNA (see: Bassam B. J. et al., Anal. Biochem., 196:80(1991)), have developed an improved method for silver staining which can detect DNA with a higher sensitivity and reproducibility, compared to the conventional silver staining methods where silver ion is linked to DNA molecule directly.
A primary object of the present invention is, therefore, to provide an improved method for high-sensitive silver staining of DNA molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and the other objects and features of the present invention will become apparent from the following description given in conjunction with the accompanying drawings, in which:
FIG. 1 (A) is a photograph showing a nitrocellulose membrane in which silver staining is carried out by the conventional Bassam et al's method.
FIG. 1 (B) is a photograph showing a nitrocellulose membrane in which silver staining is carried out by the present invention.
FIG. 2 (A) is a photograph showing a nitrocellulose membrane in which silver staining is carried out by Bassam et al's method.
FIGS. 2 (B), 2 (C), 2 (D) and 2 (E) are photographs showing nitrocellulose membranes in which silver staining is carried out by the invention through the treatment of 0.05% (v/v), 0.1% (v/v), 0.2% (v/v) and 0.4% (v/v) polyacrylic acid.
FIG. 3 (A) is a photograph showing an acrylamide gel electrophoresis for
REFERENCES:
patent: 5567585 (1996-10-01), Caetano-Anolles
Bassam, B. J. and Caetano-Anolles, G., Silver Staining of DNA in Polyacrylamide Gels, 42:181-188 (1993).
Bassam, B. J. et al., Fast and Sensitive Silver Staining of DNA in Polyacrylamide Gels, 196:80-83 (1991).
Beidler, J. L. et al., Ultrasensitive Staining of Nucleic Acids with Silver, 126:374-380 (1982).
Gottlieb, M. and Chavko, M., Silver Staining of Native and Denatured Eucaryotic DNA in Agarose Gels, 165:33-37 (1987).
Merril, C. R. et al., Coloration of Silver-Stained Protein Bands in Polyacrylamide Gels is Caused by Light Scattering from Silver Grains of Characteristic Sizes, 85:453-457 (1988).
Somerville, L. L. and Wang, Kuan., The Ultrasensitive Silver "Protein" Stain Also Detects Nanograms of Nucleic Acids, 102(1):53-58 (1981).
Switzer, III, R. C. et al., A Highly Sensitive Silver Stain for Detecting Proteins and Peptides in Polyacrylamide Gels, 98:231-237 (1979).
Wray, W., et al., Silver Staining of Proteins in Polyacrylamide Gels, 118:197-203 (1981).
Kim Jae-Jong
Park Han-Oh
You Jae-Hyung
Bioneer Corporation
Yucel Remy
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