WO2001068916A1

WO2001068916A1 - Method for detecting mutation of nucleic acid

Info

Publication number: WO2001068916A1
Application number: PCT/KR2000/000223
Authority: WO
Inventors: Gi Young Jang
Original assignee: Bionex, Inc.
Priority date: 2000-03-16
Filing date: 2000-03-16
Publication date: 2001-09-20
Also published as: AU3460600A

Abstract

Disclosed is a method for detecting mutation of nucleic acid which comprises the steps of (a) preparing a nucleic acid which has a possibility of carrying a mutation (b) adding multi-kinds of oligonucleotides, 5' end of which are phosphorylated and which are complementary to predetermined sequence of the nucleic acid (c) hybridizing the oligonucleotides to the nucleic acid (d) ligating the oligonucleotides which are adjacently hybridized to the nucleic acid using DNA ligase (e) denaturing a ligation product from the nucleic acid (f) detecting a partially ligated product. The method of the invention provides cost effective, reliable and efficient method for detecting mutation of nucleic acid.

Description

METHOD FOR DETECTING MUTATION OF NUCLEIC ACID

BACKGROUND OF THE INVENTION

The invention relates to method for mutation detection. Mutation detection or detection of sequence variation in predetermined nucleic acid has been more and more important due to the progress of the Human Genome Project. Analysis of DNA mutation or DNA sequence variation is an important source of information for finding genes involved in biological process such as reproduction, development, aging and disease. Also, detecting mutation can be applied to the analysis of disease and diagnostic, therapeutic, and preventative strategies. DNA sequencing such as dideoxy termination method of Sanger (Sanger, et al.,

Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1997) has been traditional and still most reliable method for detecting mutation. The conventional DNA sequencing method, however, needs high-resolution gel electrophoresis to separate one base difference between DNA fragments, which requires time and labor intensive procedure. To conquer the defect, new methods were proposed such as Single Stranded

Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE) and Enzyme Mismatch Cleavage (EMC) (Babon, et al., Nucleic Acids Research 23:5082-5084,1995). However, none of the foregoing methods provides a complete solution that is fast, reliable and efficient. Recently, Gene chip technologies have been developed using the conception of

DNA hybridization with high number of short nucleotides on small size of chip or other material. (DeRisi, et al., Science. 278(5338): 680-6, 1997, Wang, Science. 280: 1077- 1082, 1998.) Gene chip technologies seems promising to increase the efficiency of mutation detection but does not still be cost effective since making gene chips is complicated and expensive. Oligonucleotide Ligation Assay (OLA) and Ligation Chain Reaction (LCR) have been proposed for detection of point mutations using the fact that oligonucleotides which is hybridized adjacently to the proper orientation of nucleic acid can be covalently linked with T4 ligase (Langer, et al., Science. 241;1077, 1988) or thermostable DNA ligase (Barany, Francis PCR Methods and Applications, 1;5- 16, and U.S. Pat. No. 5,494,810, 1991). These two methods are reliable and cost effective but the detection area is limited to a couple of bases which are at jointing area of two oligonucleotides due to nature of DNA ligase.

SUMMARY OF THE INVENTION

Therefore, it is an object of the invention to overcome the problems encountered in the prior art and to provide cost effective, reliable and efficient method for detecting mutation of nucleic acid.

We found that the above object is achieved by a method for detecting mutation of nucleic acid comprising steps of: (a) preparing a nucleic acid which has a possibility of carrying a mutation;

(b) adding multi-kinds of oligonucleotides, 5' end of which are phosphorylated and which are complementary to predetermined sequence of the nucleic acid ;

(c) hybridizing the oligonucleotides to the nucleic acid; (d) ligating the oligonucleotides which are adjacently hybridized to the nucleic acid using a DNA ligase;

(e) denaturing a ligation product from the nucleic acid; and

(f) detecting a partially ligated product.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will be apparent from the following detailed description of the preferred embodiment of the invention in conjunction with the accompanying drawings, in which:

Figs. 1A-1D are schematic drawings illustrating sequential steps of making ligation product with wild type nucleic acid;

Figs. 2A-2D are schematic drawings illustrating sequential steps of making ligation product with mutant nucleic acid;

Fig. 3 is a schematic drawing illustrating base-shifted manner in which oligonucleotides are prepared; and Fig. 4 is a result of electrophoresis according to Example 1.

DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the present invention, we can detect mutation in the predetermined sequence of nucleic acid. The predetermined sequence of the nucleic acid could consist of 8-2000 nucleotides. The nucleic acid may be a part of a larger DNA, such as plasmid, a genome or DNA fragment amplified by polymerase chain reaction (PCR). The multi- kinds of oligonucleotides consisting of 4-12 nucleotides are uniform in their length and complementary to the predetermined sequence of the nucleic acid in turns. 5' end of the nucleotides are phosphorylated for ligation of DNA ligase. The kinds of oligonucleotides are determined by the nucleotide number of predetermined sequence. For example, if the predetermined sequence of the nucleic acid consists of 100 nucleotides and the oligonucleotides 5 nucleotides, the maximum number of different oligonucleotides is 20. If there are oligonucleotides having the same sequence, the number of oligonucleotides will be less than 20.

At least one oligonucleotide could carry detectable mark, such as fluorescent dye, radioisotope, digoxigenin, Cyber green, or biotin. The oligonucleotides are hybridized - ^■ • " V S+ fr /

to the nucleic acid and ligated one another in a suitable temperature range of 15-65 degrees centigrade by a suitable DNA ligase, such as T4 DNA ligase, Escherichia coli DNA ligase, Thermos thermophilus DNA ligase or Pyrococcus furiosus DNA ligase.

Figs. 1A-1D and Figs. 2A-2D illustrate sequential steps of making ligation product.

Figs. 1A-1D show the case in which nucleic acid dose not cany mutation. In Fig 1A, a nucleic acid 100 and 5 kinds of oligonucleotides 111, 112, 113, 114 and 115 are prepared. The oligonucleotides 111-115 are complementary to the predetermined sequence of nucleic acid 100 in turns. The oligonucleotide 111 designed to be hybridized to most 3' side of the nucleic acid 100 is detectably labeled at its 5' end. In Fig. IB, the oligonucleotides 111-115 are hybridized to the nucleic acid 100. In Fig. 1C, the hybridized oligonucleotides 111-115 are ligated by DNA ligase. The ligation concurrently occurs throughout the joint of five hybridized oligonucleotides 111-115. In Fig. ID, the ligation product 120 is denatured. Figs. 2A-2D show the case in which nucleic acid carries mutation. In Fig 2A, a nucleic acid 200 and 5 kinds of oligonucleotides 211, 212, 213, 214 and 215 are prepared by the same method in Fig. 1A but the nucleic acid 200 carries a mutation expressed as 'T'. In Fig. 2B, the oligonucleotides 211-215 are hybridized to the nucleic acid 200. In Fig. 2C, the hybridized oligonucleotides 211-213 are ligated one another by DNA ligase. The oligonucleotides 214 and 215, however, are not ligated because there is mismath between 'C base of the oligonucleotide 214 and mutated base 'T' of the target DNA 200. In Fig. 2D, the ligation product 220 is denatured.

The steps in Figs. IB-ID and 2B-2D could be repeated to increase total amount of ligation products. However, it can be applied when thermostable DNA ligase is used. To find the area of mutation, the ligation products 120 and 220 should be analyzed.

The ligated product 120 from the wild type nucleic acid 100 and the partially ligated product 220 from the mutant nucleic acid 200 differ in their length by two oligonucleotides. The smaller size of the ligated product 220 is easily distinguished from the full size of ligated product 120 in the gel or capillary electrophoresis or other sizing detection method. By analyzing the length of partially ligated product 220, we can detect the area of mutation in the nucleic acid 200.

According to the method in Figs. 2A-2D, we can detect only to the extent of mutations which correspond to the end bases of oligonucleotides. To detect another mutations, we should prepare another set of oligonucleotides in base-shifted manner. By adjusting base-shifted oligonucleotides several times, we can achieve complete analysis of a predetermined sequence of nucleic acid. The shifted base could be one or more.

Fig. 3 illustrates one base-shifted manner. The nucleotide number of oligonucleotides is 5 and the oligonucleotides are base-shifted 5 times. By performing mutation detection using the oligonucleotides 311-315, 321-325, 331-335, 341-345 and 351-355 prepared in the above manner, we can check up all nucleotides of predetermined sequence of nucleic acid 300 and detect exact location of mutation.

EXAMPLE

BRCA1 is breast cancer-related gene and predetermined as shown in SEQ ID NO. 1. Three different samples of BRCA1 gene from three peoples were amplified with two PCR primers of SEQ ID NOS. 2 and 3. The amplified products were cloned using convention cloning method described at Maniatis, et al., (1989, Molecular Cloning, Cold Spring Harbor Laboratory Press). Each cloned DNA product was added in three separate reaction solutions containing 15.0mM(NH₄) SO₄, 60mM Tris-HCl, pH 8.8, 6.0mM MgCl₂ and lO.OmM dithiothreital. T4 DNA liagase, a 5' IR dye-labeled oligonucleotides of SEQ ID NO. 4 and 9 different oligonucleotides of SEQ ID NOS.

5-13 which are prepared according to the predetermined sequence of BRCA1 was added also.

This reaction solution was incubated at 37 degrees centigrade for 1 hour. After addition of stopping buffer containing 95% formamide and 0. 1% Brumophenol Blue, that was added to automated DNA sequencer LI-COR 4200, and analyzed by electrophoresis.

The result of electrophoresis was shown in Fig. 4. The mutation area of mutant DNA (G-T conversion at 96^th base of SEQ TD NO. 1) in lane 3 and 4 were detected compared to the wild type DNA in first two lanes.

Although the preferred embodiments of the invention have been disclosed for illustrative purpose, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

What is claimed is:

1. A method for detecting mutation of nucleic acid, comprising the steps of:

(a) preparing a nucleic acid which has a possibility of carrying a mutation;

(b) adding multi-kinds of oligonucleotides, 5' end of which are phosphorylated and which are complementary to the predetermined sequence of the nucleic acid;

(c) hybridizing the oligonucleotides to the nucleic acid;

(d) ligating the oligonucleotides which are adjacently hybridized to the nucleic acid using a DNA ligase; (e) denaturing a ligation product from the nucleic acid;

(f) detecting a partially ligated product.

2. The method for detecting mutation of nucleic acid according to claim 1, wherein the steps (c) through (e) is repeated at least one time.

3. The method for detecting mutation of nucleic acid according to claim 1, wherein the DNA ligase is selected from the group consisting of T4 DNA ligase,

Escherichia coli DNA ligase, Thermos thermophilus DNA ligase and pyrococcus furiousus DNA ligase.

4. The method for detecting mutation of nucleic acid according to claim 1, wherein at least one oligonucleotide of multi-kinds of oligonucleotides is detectably labeled.

5. The method for detecting mutation of nucleic acid according to claim 1, further comprises that steps (a) through (f) are repeated at least one times under the condition that the oligonucleotides are prepared in one or more bases-shifted manner on the basis of the predetermined sequence of the nucleic acid.