A METHOD FOR HIGH-SENSITIVE SILVER STAINING

Abstract: The present invention provides 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. The silver staining method of the present invention can be applied for the detection and sequencing of trace amount of DNA.

Description: A METHOD FOR HIGH-SENSITIVE SILVER STAINING BACKGROUND OF THE INVENTION 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. 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 followings: 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 stainig methods for the detection of DNA have been developed as followings: 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, R. , 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 stainig 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: Figure 1 (A) is a photograph showing a nitrocellulose membrane in which silver staining is carried out by the conventional Bassam et al's method. Figure 1 (B) is a photograph showing a nitrocellulose membrane in which silver staining is carried out by the present invention. Figure 2 (A) is a photograph showing a nitrocellulose membrane in which silver staining is - carried out by Bassam et al's method. Figures 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. Figure 3 (A) is a photograph showing an acrylamide gel electrophoresis for nucleotide sequencing in which silver staining is carried out by Bassam et al's method. Figure 3 (B) is a photograph showing an acrylamide gel electrophoresis for nucleotide sequencing in which silver staining is carried out by the invention through the treatment of 0.2% (v/v) polyacrylic acid. DETAILED DESCRIPTION OF THE INVENTION The present inventors have improved the sensitivity of Bassam et al's silver staining method for DNA detection (see : Bassam, B. J. et al . , Anal. Biochem., 196:80(1991)) which has been known to be a simple and highly sensitive method, by immersing a gel containing DNAs to an aqueous solution containing a polymer and binding silver ions to the DNA-polyτner complex. In this regard, the polymer which has a high affinity for DNA molecule, preferably polyacrylic acid or polyethyleneimine, is linked to the DNAs to induce coloring of the silver ions. The concentration of polymer in the aqueous solution (e .g. , water), though it may be varied depending on the kind of the polymer, ranges in a concentration of 0.2% (v/v) to 0.4% (v/v), if polyacrylic acid is employed. In accordance with the silver staining method of the invention, even ng of DNA can be detected with a higher reproducibility, compared to Bassam et al's method which detects μg of DNA, when DNA is spotted in a round shape of 5mm in diameter on a nitrocellulose membrane. The silver staining method of the invention can be applied for nucleotide sequencing and detecting traces of DNA molecules. The present invention is further illustrated by the following examples, which should not be taken to limit the scope of the invention. Example 1 : Effect of Polyacrylic Acid on the Sensitivity of Silver Staining In order to investigate the effect of polyacrylic acid on the sensitivity of silver staining of DNA, λ DNA diluted to a concentration was spotted in a round shape of 5mm in diameter on a nitrocellulose membrane. After subjecting the membrane in a drying oven maintained at a temparature of 65°C for lhr, it was soaked in 0.2% (v/v) polyacrylic acid solution for lOmin. Then, silver staining was performed in accordance with Bassam et al's method (see: Bassam et el., Anal. Biochem., 196:80(1991)): Silver ion was bound to polyacrylic acid by treating the nitrocellulose membrane with polyacrylic acid and soaking in an aqueous silver containing lg/L of silver nitrate solution and 1.5ml/L formaldehyde for 20min. The- above nitrocellulose membrane was rinsed with distilled water for 5 sec to eliminate unbound silver ion and subjected in a developing solution containing 30g/L of sodium carbonate, 3ml/L of formaldehyde and 2mg/L of sodium thiosulfate. Then, 7.5% acetic acid was added to stop the reaction when color is sufficiently developed. On the other hand, DNA sample stained with silver nitrate according to Bassam et al's method was used as a control. Fig. 1(A) shows a nitrocellulose membrane stained with silver nitrate by Bassam et al's method, which has been reported as the best silver staining method so far. Fig. 1(B) shows a nitrocellulose membrane treated with polyacrylic acid by the invented method. In Figs. 1(A) and 1(B), lanes 1-9 contain lμg, 500ng, 250ng, 125ng, 60ng, 40ng, 2ng, lng and lOOpg of λ DNA, respectively. As shown in Figs., 1(A) and 1(B), the invented method which employs polyacrylic acid can detect lng of DNA with a higher sensitivity, compared to the conventional Bassam et al's method which can detect lμg of DNA. These results demonstrate that the newly developed silver staining method improves the sensitivity of dectection of DNA. Example 2 : Effect of Polyacrylic Acid Concentration on the Sensitivity of Silver Staining In order to investigate the optimal polyacrylic acid concentration, silver staining was performed by using 0.05% (v/v), 0.1% (v/v), 0.2% (v/v) and 0.4%(v/v) polyacrylic acid, respectively, in an analogous manner as in Example 1 (see: Figs. 2 (A) -2 (E) ) . Fig. 2(A) shows a nitrocellulose membrane in which silver staining is carried out by Bassam et al's method and Figs. 2(B) to 2(E) show nitrocellulose membranes in which silver staining is carried out through the treatment with 0.05% (v/v), 0.1% (v/v), 0.2% (v/v) and 0.4% (v/v) polyacrylic acid, respectively. In Figs. 2(A) to 2(E), lanes 1-9 contain l g, 500ng, 250ng, 125ng, 60ng, 40ng, 12ng, lng and lOOpg of λ DNA, respectively. As shown in Figs. 2(B) to 2(E), the sensitivity increased with the concentration of polyacrylic acid, while background signal also increased at a concentration above 0.4% (v/v). Accordingly, it was determined that the optimal concentration of polyacrylic acid for silver staining is about 0.2% (v/v). Example 3 : Silver Staining of DNA Sequencing Gel In order to investigate the effect of polyacrylic acid on the sensitivity of silver staining in DNA sequencing polyacrylamide gel, DNA sequencing reactions were performed by using 200ng, lOOng, 40ng, 20ng and lOng of template DNA, respectively, which was separated on 6% polyacrylamide gel electrophoresis and stained with silver nitrate . The DNA sequencing reactions were performed by using Top™ DNA sequencing kit (Bioneer Corporation, Korea) as followings: First, the template DN (497 base pair PCR product) was mixed with 30 pmoles of primer (5'- GTTTTGGCTTCCAGTTCCACC-3' ) , and the final volume was adjusted to 40 tf with distilled water. Then, lOμi aliquots of the samples were added to four separate reaction tubes containing Top™ DNA polymerase, 50mM Tris-HCl buffer (containing 2mM MgCl 2 , pH 8.3) and G(20μM of ddGTP/6μM dNTP) , A(170μM of ddATP/6μM dNTP) , T(300μM of ddTTP/6μM dNTP) and C(200 M of ddCTP/6μM dNTP), respectively. 40 rounds of PCR cycling was carried out as followings: 94°C for 5 min (predenaturation) , 94°C for 30 sec (denaturaion) , 55°C for 30 sec (annealing) , and 72°C for 1 min (extension) . At the end of the amplification reaction, 4μβ of stop solution containing 98% formamide, lOmM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue was added to each reaction tube. After heating for 5min at 95°C, each reaction was analysed on a 6% polyacrylamide gel . Figs. 3(A) and 3(B) show the results from silver staining of the DNA sequencing polyacrylamide gel by Bassam et al's method and the invented method after treatment with 0.2% (v/v) polyacrylic acid. In Figs. 3(A) and 3(B), lanes 1-5 contain 200ng, lOOng, 40ng, 20ng, and lOng of the template DNA, respectively. As shown in Figs. 3(A) and 3(B), in contrast with the conventional Bassam et al's method which can detect DNA of lOOng level, the present method accompanying polyacrylic acid ' treatment can detect DNA even when lOng of template DNA was employed. Accordingly, it was clearly determined that the invented silver staining method can also be available for DNA sequencing with a high sensitivity and reproducibility. As clearly illustrated and demonstrated as aboves, the present invention provides an improved method for high-sensitive silver staining of DNA molecules with a high sensitivity, which can detect even ng level of DNA molecules. Accordingly, the silver staining method of the invention can be applied for the detection and sequencing of trace amount of DNA. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BIONEER CORPORATION et al . (B) STREET: 124, Cheokbuk-Ri, Namee-Myun (C) CITY: Cheongwon-Kun, Choongcheongbuk-Do (D) STATE: not applicable (E) COUNTRY: Korea (F) POSTAL CODE (ZIP) : 363-810 (G) TELEPHONE: 0431-60-6060 (H) TELEFAX: 0431-60-2009 (ii) TITLE OF INVENTION: A method for high-sensitive silver staining (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vii) IMMEDIATE SOURCE: (B) CLONE: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 GTTTTGGCTT CCAGTTCCAC C

Claims: WHAT IS CLAIMED IS: 1. A method for high-sensitive silver stainig which comprises the steps of: linking DNA to a polymer which has a high affinity for DNA, to give a DNA-polymer complex by immersing a DNA containing gel to an aqueous solution containing the polymer; and, binding silver ions to the DNA-polymer complex. 2. The method for high-sensitive silver staining of claim 1, wherein the polymer is polyacrylic acid or polyethyleneimine . 3. The method for high-sensitive silver staining of claim 1, wherein the solvent employed in the aqueous solution is water. 4. The method for high-sensitive silver staining of claim 1, wherein the concentration of the polymer in the aqueous solution ranges 0.2% (v/v) to 0.4% (v/v). 5. The method for high-sensitive silver staining of claim 1, wherein the DNA containing gel is for DNA sequencing. 6. The method for high-sensitive silver staining of claim 1, wherein the DNA containing gel is for DNA detection.

Inventors: KIM JAE-JONG, PARK HAN-OH, YOU JAE-HYUNG, KIM, JAE-JONG, PARK, HAN-OH, YOU, JAE-HYUNG
Applicants: BIONEER CORP, KIM JAE JONG, PARK HAN OH, YOU JAE HYUNG, BIONEER CORPORATION, KIM, JAE-JONG, PARK, HAN-OH, YOU, JAE-HYUNG
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1, G01N33