WO2002004673A1

WO2002004673A1 - Method for detecting sequence variation of nucleic acid

Info

Publication number: WO2002004673A1
Application number: PCT/KR2000/000753
Authority: WO
Inventors: Gi Young Jang
Original assignee: Bionex, Inc.
Priority date: 2000-07-12
Filing date: 2000-07-12
Publication date: 2002-01-17
Also published as: AU2000257122A1

Abstract

The present invention provides a method for detection of sequence variation of nucleic acid comprising steps of (a) preparing a nucleic acid which has a possibility of carrying sequence variation, (b) adding two or more different oligonucleotides, which are complementary to the nucleic acid, 5' end of which are different detectable marker-labeled and 3' end of which have sequence complementary to two or more different predetermined sequence variants of the nucleic acid respectively, (c) denaturing the nucleic acid by heating, (d) hybridizing the oligonucleotides to the nucleic acid and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually, and (e) detecting a extended nucleic acid product. The present invention provides simple and convenient method for sequence variations detection.

Description

METHOD FOR DETECTING SEQUENCE VARIATION OF

NUCLEIC ACID

BACKGROUND OF THE INVENTION

The invention relates to method for sequence variation of nucleic acid. 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 sequence variation of nucleic acid is an important source of information for finding genes involved in biological process such as reproduction, development, aging and disease. Also, detecting sequence variation of DNA 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 sequence variation. 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.

For improvement, new alternative 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.

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,l;5-16, and U.S. Pat. No. 5.494.810. 1991). These two methods are reliable and cost effective but it isn't easy to adapt the ligase-based technology for high throughput data collection due to nature of DNA ligase. 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, et al, Science. 280:1077-1082, 19983) Gene chip technologies seem promising to increase the efficiency of sequence variation of DNA but does not still reliable due to characteristic of hybridization between probe and DNA strand.

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 sequence variation of nucleic acid. We found that the above object is achieved by a method for detecting sequence variation ofnucleic acid comprising steps of:

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

(b) adding two or more different oligonucleotides, which are complementary to the nucleic acid, 5' end of which are different detectable marker-labeled, and 3' end of which have sequence complementary to two or more different predetermined sequence variants of the nucleic acid respectively;

(c) denaturing the nucleic acid by heating;

(d) hybridizing the oligonucleotides to the nucleic acid and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually; and (e) detecting a extended nucleic acid product.

Another aspect of present invention provides a method for detecting sequence variation of nucleic acid comprising steps of: (a) preparing one or more nucleic acids which have a possibility of carrying plural kinds of sequence variations;

(b) adding plural set of two or more different oligonucleotides, which are complementary to one or more nucleic acid, 5' end of which are different detectable marker-labeled, and 3' end of which have sequence complementary to plural kind of two or more different predetermined sequence variants of the nucleic acids respectively;

(c) denaturing the nucleic acids by heating;

(d) hybridizing the oligonucleotides to the nucleic acid respectively and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually; and

(e) detecting a extended nucleic acid product.

The other aspect of present invention provides a method for detecting sequence variation of nucleic acid comprising steps of:

(a) preparing a nucleic acids of double strand which have a possibility of carrying sequence variation;

(b) adding two or more different universal oligonucleotides which are not complementary to any sequence of the nucleic acid and 5' end of which are different detectable maker-labeled;

(c) adding two or more different oligonucleotides, which are complementary to one strand of the nucleic acid, 5' end of which have tail of the same sequence with the universal oligonucleotides in their 5' end, and 3' end of which have sequence complementary to the different predetermined sequence variants of the nucleic acid, respectively;

(d) adding standard PCR oligonucleotides which are complementary to the other strands of the nucleic acid;

(e) denaturing the nucleic acid by heating;

(f) hybridizing the oligonucleotides to the nucleic acid and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually; and (g) detecting a extended nucleic acid products of the universal oligonucleotides.

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 detecting nucleic acid which contains homozygotic base "G";

Figs. 2A-2D are schematic drawings illustrating sequential steps of detecting nucleic acid which contains homozygotic base "A";

Figs. 3A-3D are schematic drawings illustrating sequential steps of detecting nucleic acid which contains heterozygotic base "G" and "A";

Figs. 4A-4D are schematic drawings illustrating sequential steps of detecting sequence variation by using universal labeled dyes; and

Fig. 5 is a result of electrophoresis according to Example 1. DESCRIPTION OF THE PREFERRED EMBODEMENT

According to the present invention, we can detect sequence variation of the predetermined sequence of nucleic acid using two or more different oligonucleotides more efficiently. The predetermined sequence of the nucleic acid could consist of 30- 200,000 nucleotides. The nucleic acid may be DNA fragment amplified by Polymerase Chain Reaction (PCR) or a part of a larger DNA, such as plasmid, a genome. Two or more different oligonucleotides may consist of 7-20 nucleotides and be uniform in their length. They are designed to be complementary to the predetermined sequence of the nucleic acid. Especially, their 3' ends have sequence complementary to the predetermined variants of the nucleic acid. Each oligonucleotide carries different kinds of detectable marker, such as fluorescent dye, radioisotope, digoxigenin, Cyber green, or biotin.

During the process of sequential steps, the oligonucleotides are competitively hybridized to the nucleic acid and extended. To differentiate the hybridization of perfectly matched and mismatched oligonucleotides, hybridization temperature is gradually decreased. After extending process using a suitable DNA polymerase such as Taq DNA polymerase, the extension products are detected. By ascertaining the kind of detectable mark, we can detect the sequence variation of the nucleic acid.

Figs. 1A-1D illustrate sequential steps of detecting nucleic acid which carries homozygotic base "G".

In Fig 1A, a nucleic acid 110 carries a homozygotic base "G". To detect the sequence variation of the nucleic acid 110, 2 kinds of oligonucleotides 130 and 140 are prepared which are complementary to the previously known variation region of the nucleic acid. 5' ends of the oligonucleotides 130 and 140 are different detectable marker-labeled and 3' ends are respectively complementary to two different known sequence variant of the nucleic acid, "G" and "A". Two different detectable markers at 5' ends of oligonucleotides 130 and 140 are used to differentiate two extension products easily. For example, two fluorescent dyes of different detection wave lengths can be attached at 5' ends of oligonucleotides 130 and 140.

In Fig. IB, the oligonucleotides 130 and 140 are hybridized to the nucleic acid 110. Because the nucleic acid 110 carries homozygotic base "G", the oligonucleotide 130 is matched perfectly to the nucleic acid 110 and is hybridized to the nucleic acid 110 more easily than the oligonucleotide 140. Though the oligonucleotide 140 is hybridized to the nucleic acid 100, the extension of oligonucleotides 140 will not efficiently occur due to the mismatched base at its 3 'end. To improve differential hybridization between two oligonucleotides 130 and 140, the temperature condition of hybridization is gradually decreased. Desirably, the temperature starts from 40-65 ^°C and is ramped down to 20-39 ^°C with cooling rate of 0.01-3 ^°C per second. Also the temperature condition can start from 35 -65 ^°C and be cooled down to 20-34 ^°C with gradual step-down of 0.1-4 °C. For example, if the temperature condition is decreased from 55 °C to 27 °C, the temperature is maintained 55 °C for lmin, then 54 °C for lmin and cooled gradually down up to 27 °C in this method. In the case of ramping-down hybridization, the first hybridizing temperature is maintained 55^°C for lmin, then ramped down to 27 °C with cooling rate of 0.02 °C per second.

Normally the annealing temperature of perfectly matched oligonucleotide 130 is higher than that of mismatched oligonucleotide 140. The difference of anneal temperature between matched and mismatched heteroduplex complex is more than one degree, typically two or three degree. If the hybridizing temperature is gradually decreased from higher temperature than the annealing temperature of perfectly matched oligonucleotide 130 to lower temperature than the annealing temperature of mismatched oligonucleotide 140, the perfectly matched oligonucleotide 130 will occupy most of target region of the nucleic acid 110 first.

In Fig. 1C, the hybridized oligonucleotide 130 is extended. In Fig. ID, the extension product 131 can be detected using, for example, automated DNA sequencer, gel scanner or Microarray scanner. By ascertaining the kind of detectable marker combined in the extension product 131, the sequence variation, "C" can be distinguished.

Figs. 2A-2D illustrate sequential steps of detecting nucleic acid which carries homozygotic base "A".

Two kinds of oligonucleotides 230 and 240 are prepared by the same method of Fig. 1A but the nucleic acid 220 carries a homozygotic base "A". In Fig.2B, the oligonucleotides 230 and 240 are hybridized to the nucleic acid 220 in the ramping down temperature condition. In Fig. 2C, the hybridized oligonucleotide 240 is extended by DNA polymerase. The oligonucleotide 230, however, is not efficiently extended because there is mismatch between "C" base of the oligonucleotide 230 and "A" base of the nucleic acid 220. In Fig. 2D, the extension product 241 is detected. By detecting the extension product 241 and ascertaining the kind of detectable marker, we can distinguish the sequence variation of the nucleic acid 220.

Figs. 3A-3D show the case in which nucleic acid carries heterozygotic base. In

Fig 3A, 2 kinds of oligonucleotides 330, 340 are prepared by the same method of Fig. 1A.

In Fig 3B, the oligonucleotides 330 and 340 are respectively hybridized to the nucleic acid 310 and 320 in ramping-down or stepping down temperature condition, and extended by DNA polymerase in Fig. 3C.

In Fig. 3D, the extended DNA fragment is detected. In the process of ascertaining the kind of detectable marker combined in extension product, two kinds of markers are found and the nucleic acid is distinguished to have heterozygotic base "G" and "A".

The steps in Figs. IB-ID, 2B-2D and 3B-3D could be repeated respectively to increase total amount of extension products when thermostable DNA polymerase is used.

The extended DNA fragment could be analyzed by Automated DNA sequencer, gel scanner or Microarray scanner, which detect the signal from the detectable markers. To facilitate the isolation of extended DNA fragments, biotin-bound dUTP can be added into dNTP solution or substituted for dTTP during the extension by DNA polymerase. Therefore, the extended DNA fragment containing biotin-bound dUTP, can be easily manipulated using straptoavidin-coated solid material such as glass plate, magnetic bead, nylon membrane, paper, and plastic. The application of biotin-bound dUTP with straptoavidin-coated solid material allows for high throughput automated detection of extended DNA fragment using DNA chip scanner and other detecting instruments.

For high throughput detection of sequence variation, plural target nucleic acid are amplified by using two oligonucleotides which consist of normal primer and 5'biotin bound primer or normal primer and 5'amine-bound primer separately. Then the amplified nucleic acids are spotted respectively on the streptoavidin-coated or aldehyde-coated solid material such as glass plate, nylon membrane, paper and plastic. During hybridizing and extending process, the selective extension of two different detectable marker-labeled oligonucleotides occurs on the surface of solid material. After stingency washing step is performed to remove un-extended oligonucleotides, the extension products which are combined to the nucleic acid which are spotted on solid material can be detected at one time, so the throughput efficiency of detection increases.

This method also can be used in detecting plural kind of sequence variations of one or more target nucleic acid. To detect plural kinds of sequence variation of nucleic acid, plural set of oligonucleotides that are designed to be complementary to the plural kind of two or more predetermined sequence variants respectively, should be prepared. Each set of oligonucleotide carries different kinds of detectable marker. The plural set of oligonucleotides are hybridized to each variation region of the nucleic acids and extended in a suitable ramping temperature. By detecting the extension products of plural set of oligonucleotides, we can distinguish plural kind of sequence variations.

Figs. 4A-4D illustrate detection of sequence variation using tailed oligonucleotides and universal labeled oligonucleotides. This method allows us to avoid making new labeled oligonucleotides and to reduce the detection cost.

In Fig. 4A, the nucleic acids 401-404 are separated two double strand which carry heterozygotic base "G" and "A". To detect the sequence variation 5 kinds of oligonucleotides 411-415 are added. The oligonucleotide 411, 412 and 413 are PCR primers which complementary to the nucleic acid 401-404. The oligonucleotides 411 and 412 have tail parts in their 5' side, which have the same sequence of oligonucleotides 414 and 415 respectively and 3' side of them have sequence complementary to the sequence variation of the nucleic acid 402 and 404. The oligonucleotide 413 is standard PCR primer which is complementary to the nucleic acid 401 and 403. The universal oligonucleotides 414 and 415 are differently labeled each other. The size of tail part in tailed oligonucleotide, 410 and 411 can be from 8 to 20 bases and the size of labeled oligonucleotide can be from 8 to 20 bases. In Fig. 4B, the oligonucleotide 411, 412 and 413 are hybridized to the double strand of DNA and extended. After these processes are repeated as shown in Fig. 4C, there appear extension products 411^* and 412^*, 5' side parts of which are complementary to the universal labeled oligonucleotides 414 and 415 respectively, so the oligonucleotides 414 and 415 are hybridized to the extension products 411^* and 412^* and extended as shown in Fig. 4D. By detecting the extension products of the oligonucleotides 414 and 415, we can distinguish the sequence variation of nucleic acid.

Example 1

LIPC is Lypoprotein gene and predetermined as shown in SEQ ID NO.l. Thirty two sample of LIPC gene from thirty two peoples were amplified with two PCR primer of SEQ TD NO.2 and 3 respectively. The amplified products were purified to remove excess of primers using conventional alcohol precipitation method described at

Maniatis, et al (1989, Molecular Cloning, Cold Spring Harbor Laboratory press). The sequence variation of "T" and "C" base appears at 130^th base of SEQ ID NO.l and two IRD700 and IRD800 dye-labeled oligonucleotides are shown in SEQ H) NO.4 and 5.

Each purified DNA product was added in a reaction solution containing lOmM

Tris-Hcl, pH8.8, 2.0mM Mgcl2, 50mM Kcl and 200mM of dNTP. Taq DNA polymerase, a 5' IR700 dye-labeled oligonucleotide and 5' IR800 dye-labeled oligonucleotie were added also. This reaction solution was inbutated at the ramping-down condition from 55 °C to

37 °C for lhr. After addition of stopping buffer containing 95% formamide and 0.1%.

Brumophenol Blue, that was loaded to automated DMA Sequencer LI-COR 4200 (LI-

COR, LINCOLN, NE, USA) and analyzed by electrophoresis.

The result of electrophoresis is shown in Fig. 5. The signal at both IRD700 and IRD 800 represents heterozygotic base "T/C" while the signal at only IRD 700 and IRD 800 represents homozygotic base "T" and "C" respectively.

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 sequence variation of nucleic acid, comprising the steps of:

(a) preparing a nucleic acid which has a possibility of carrying sequence variation; (b) adding two or more different oligonucleotides, which are complementary to the nucleic acid, 5' end of which are different detectable marker-labeled and 3' end of which have sequence complementary to two or more different predetermined sequence variants of the nucleic acid respectively; (c) denaturing the nucleic acid by heating; (d) hybridizing the oligonucleotides to the nucleic acid and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually; and (e) detecting a extended nucleic acid products.

2. A method according to claim 1, wherein the steps (c) through (d) are repeated at least one time.

3. A method according to claim 1, wherein the nucleic acid is prepared by Polymerase Chain Reaction with two oligonucleotides primers which are selected respectively from the group consisting of a normal unmodified oligonucleotide primer, 5' biotin-labeled oligonucleotide primer and 5'amine-labeled oligonucleotide primer.

4. A method according to claim 3, wherein the nucleic acid is immobilized a solid material selected from the group consisting of glass plate, membrane and magnetic bead.

5. A method according to claim 1, wherein the nucleic acid is prepared by cutting cloned DNA using restriction enzyme. 6. A method according to claim 1, wherein the oligonucleotides consist of 7-20 nucleotides.

7. A method according to claim 1, wherein the oligonucleotides are uniform in their length.

8. A method according to claim 1, wherein the different detectable markers are fluorescent dyes.

9. A method according to claim 1, wherein the temperature condition of step (d) starts from 40-65 ^°C and is ramped down to 20-39 ^°C with cooling rate of 0.01-3 ^°C degree per second.

10. A method according to claim 1, wherein the temperature condition starts from 35-65 ^°C and is cooled down to 20-34 ^°C with gradual step-down of 0.1-4 ^°C .

11. A method according to claim 1, wherein the extension occurs by thermostable enzyme using four different nucleoside triphosphates, dATP, dGTP, dCTP and biotin- bound dUTP.

12. A method according to claim 1, wherein the detection of extension products is achieved by using automated DNA sequencer, gel scanner or Microarray scanner.

13. A method for detecting sequence variation of nucleic acid, comprising the steps of:

(a) preparing one or more nucleic acids which have a possibility of carrying plural kinds of sequence variations; (b) adding plural set of two or more different oligonucleotides, which are complementary to one or more nucleic acid, 5' end of which are different detectable marker-labeled, and 3' end of which have sequence complementary to plural kind of two or more different predetermined sequence variants of the nucleic acids respectively; (c) denaturing the nucleic acids by heating; (d) hybridizing the oligonucleotides to the nucleic acid respectively and extending using thermostable DNA polymerase on the condition of decreasing the temperature gradually; and

(e) detecting a extended nucleic acid products. 14. A method according to claim 15, wherein the steps (c) through (d) are repeated at least one time.

15. A method according to claim 15, wherein the nucleic acids are prepared by Polymerase Chain Reaction with two oligonucleotides primers which are selected respectively from the group consisting of a normal unmodified oligonucleotide primer, 5' biotin-labeled oligonucleotide primer and 5'amine-labeled oligonucleotide primer.

16. A method according to claim 17, wherein the nucleic acids are immobilized a solid material selected from the group consisting of glass plate, membrane and magnetic bead.

17. A method according to claim 15, wherein the nucleic acids are prepared by cutting cloned DNA using restriction enzyme.

18. A method according to claim 15, wherein the plural set of oligonucleotides consist of 7-20 nucleotides.

19. A method according to claim 15, wherein the oligonucleotides are uniform in their length. 20. A method according to claim 15, wherein the different detectable markers are fluorescent dyes.

21. A method according to claim 15, wherein the temperature condition of step (d) starts from 40-65 ^°C and is ramped down to 20-39 ^°C with cooling rate of 0.01-3 ^°C degree per second. 22. A method according to claim 15, wherein the temperature condition starts from 35-65 ^°C and is cooled down to 20-34 ^°C with gradual step-down of 0.1-4 ^°C .

23. A method according to claim 15, wherein the extension occurs by thermostable enzyme using four different nucleoside triphosphates, dATP, dGTP, dCTP and biotin-bound dUTP 24. A method according to claim 15, wherein the detection of extension products is achieved by using automated DNA sequencer, gel scanner or Microarray scanner.

25. A method for detecting sequence variation of nucleic acid, comprising the steps of:

(a) preparing a nucleic acid of double strand which have a possibility of carrying sequence variation;

(c) adding two or more different oligonucleotides, which consist of two parts of sequence, 5' side part of which have tail of the same sequence with the universal oligonucleotides, and 3' side part of which have sequence complementary to the different predetermined sequence variants of the nucleic acid respectively;

(e) denaturing the nucleic acid by heating;

26. A method according to claim 29, wherein the steps (e) through (f) are repeated at least two time.

27. A method according to claim 29, wherein the nucleic acid is prepared by Polymerase Chain Reaction with two oligonucleotides primers which are selected respectively from the group consisting of a normal unmodified oligonucleotide primer, 5' biotin-labeled oligonucleotide primer and 5'amine-labeled oligonucleotide primer.

28. A method according to claim 29, wherein the nucleic acid is immobilized a solid material selected from the group consisting of glass plate, membrane and magnetic bead. 29. A method according to claim 29, wherein the nucleic acid is prepared by cutting cloned DNA using restriction enzyme.

30. A method according to claim 29, wherein the oligonucleotides consist of 7-20 nucleotides.

31. A method according to claim 29, wherein the oligonucleotides are uniform in their length.

32. A method according to claim 29, wherein the different detectable markers are fluorescent dyes.

33. A method according to claim 29, wherein the temperature condition of step (d) starts from 40-65 ^°C and is ramped down to 20-39 ^°C with cooling rate of 0.01-3 ^°C degree per second.

34. A method according to claim 29, wherein the temperature condition starts from 35 -65 ^°C and is cooled down to 20-34 ^°C with gradual step-down of 0.1-4 ^°C .

35. A method according to claim 29, wherein the extension occurs by thermostable enzyme using four different nucleoside triphosphates, dATP, dGTP, dCTP and biotin-bound dUTP.

6. A method according to claim 29, wherein the detection of extension products ieved by using automated DNA sequencer, gel scanner or Microarray scanner.