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WO2002004673A1 - Procede pour detecter une variation de sequence dans un acide nucleique - Google Patents

Procede pour detecter une variation de sequence dans un acide nucleique Download PDF

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Publication number
WO2002004673A1
WO2002004673A1 PCT/KR2000/000753 KR0000753W WO0204673A1 WO 2002004673 A1 WO2002004673 A1 WO 2002004673A1 KR 0000753 W KR0000753 W KR 0000753W WO 0204673 A1 WO0204673 A1 WO 0204673A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
oligonucleotides
sequence
different
complementary
Prior art date
Application number
PCT/KR2000/000753
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English (en)
Inventor
Gi Young Jang
Original Assignee
Bionex, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bionex, Inc. filed Critical Bionex, Inc.
Priority to PCT/KR2000/000753 priority Critical patent/WO2002004673A1/fr
Priority to AU2000257122A priority patent/AU2000257122A1/en
Publication of WO2002004673A1 publication Critical patent/WO2002004673A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • 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.,
  • DGGE DGGE
  • EMC Enzyme Mismatch Cleavage
  • Oligonucleotide Ligation Assay OLA
  • Ligation Chain Reaction LCR
  • T4 ligase Longer, et al., Science. 241:1077, 1988
  • thermostable DNA ligase Barany, Francis, PCR Methods and Applications,l;5-16, and U.S. Pat. No. 5.494.810. 1991.
  • 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;
  • the other aspect of present invention provides a method for detecting sequence variation of nucleic acid comprising steps of:
  • 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.
  • Fig. 5 is a result of electrophoresis according to Example 1. DESCRIPTION OF THE PREFERRED EMBODEMENT
  • 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.
  • the oligonucleotides are competitively hybridized to the nucleic acid and extended.
  • hybridization temperature is gradually decreased.
  • 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".
  • a nucleic acid 110 carries a homozygotic base "G".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the hybridized oligonucleotide 130 is extended.
  • 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".
  • oligonucleotides 230 and 240 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".
  • the oligonucleotides 230 and 240 are hybridized to the nucleic acid 220 in the ramping down temperature condition.
  • the hybridized oligonucleotide 240 is extended by DNA polymerase.
  • the oligonucleotide 230 is not efficiently extended because there is mismatch between "C" base of the oligonucleotide 230 and "A" base of the nucleic acid 220.
  • 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.
  • Fig 3A 2 kinds of oligonucleotides 330, 340 are prepared by the same method of Fig. 1A.
  • 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.
  • Fig. 3D the extended DNA fragment is detected.
  • two kinds of markers are found and the nucleic acid is distinguished to have heterozygotic base "G" and "A".
  • the extended DNA fragment could be analyzed by Automated DNA sequencer, gel scanner or Microarray scanner, which detect the signal from the detectable markers.
  • 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.
  • straptoavidin-coated solid material such as glass plate, magnetic bead, nylon membrane, paper, and plastic.
  • 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.
  • the selective extension of two different detectable marker-labeled oligonucleotides occurs on the surface of solid material.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • extension products 411 * and 412 * 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.
  • detecting the extension products of the oligonucleotides 414 and 415 we can distinguish the sequence variation of nucleic acid.
  • 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
  • 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
  • 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.

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  • Organic Chemistry (AREA)
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  • Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé pour détecter une variation de séquence dans un acide nucléique. Ce procédé consiste (a) à préparer un acide nucléique qui peut faire l'objet d'une variation de séquence, (b) à ajouter deux ou plusieurs oligonucléotides différents, qui sont complémentaires à l'acide nucléique, dont les extrémités 5' sont marquées par marqueur de manière différemment détectable et dont les extrémités 3' présentent respectivement des séquences complémentaires à deux ou plusieurs variantes de séquence prédéfinie différentes de l'acide nucléique, (c) à dénaturer l'acide nucléique par traitement thermique, (d) à hybrider les oligonucléotides à l'acide nucléique, à réaliser un allongement, par utilisation d'une ADN polymérase thermostable, dans des conditions de diminution progressive de la température, puis (e) à détecter un produit d'acide nucléique allongé. Le procédé de détection de variation de séquence selon la présente invention est simple et pratique.
PCT/KR2000/000753 2000-07-12 2000-07-12 Procede pour detecter une variation de sequence dans un acide nucleique WO2002004673A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/KR2000/000753 WO2002004673A1 (fr) 2000-07-12 2000-07-12 Procede pour detecter une variation de sequence dans un acide nucleique
AU2000257122A AU2000257122A1 (en) 2000-07-12 2000-07-12 Method for detecting sequence variation of nucleic acid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2000/000753 WO2002004673A1 (fr) 2000-07-12 2000-07-12 Procede pour detecter une variation de sequence dans un acide nucleique

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0332435A2 (fr) * 1988-03-10 1989-09-13 Zeneca Limited Procédé pour détecter des séquences de nucléotides
US5605794A (en) * 1991-09-06 1997-02-25 Boehringer Mannheim Gmbh Method of detecting variant nucleic acids
US6013431A (en) * 1990-02-16 2000-01-11 Molecular Tool, Inc. Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators
EP1061135A2 (fr) * 1999-06-17 2000-12-20 Becton Dickinson and Company Procédés et oligonucléotides pour la détection des variations des séquences d'acides nucléiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0332435A2 (fr) * 1988-03-10 1989-09-13 Zeneca Limited Procédé pour détecter des séquences de nucléotides
US6013431A (en) * 1990-02-16 2000-01-11 Molecular Tool, Inc. Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators
US5605794A (en) * 1991-09-06 1997-02-25 Boehringer Mannheim Gmbh Method of detecting variant nucleic acids
EP1061135A2 (fr) * 1999-06-17 2000-12-20 Becton Dickinson and Company Procédés et oligonucléotides pour la détection des variations des séquences d'acides nucléiques

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Publication number Publication date
AU2000257122A1 (en) 2002-01-21

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