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WO1994017203A1 - Procede d'analyse d'empreinte d'adn amplifie permettant de detecter les variations du genome - Google Patents

Procede d'analyse d'empreinte d'adn amplifie permettant de detecter les variations du genome Download PDF

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Publication number
WO1994017203A1
WO1994017203A1 PCT/CA1994/000033 CA9400033W WO9417203A1 WO 1994017203 A1 WO1994017203 A1 WO 1994017203A1 CA 9400033 W CA9400033 W CA 9400033W WO 9417203 A1 WO9417203 A1 WO 9417203A1
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WIPO (PCT)
Prior art keywords
nucleotide sequence
accordance
strain
sequence
linker
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CA1994/000033
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English (en)
Inventor
Dennis Kunimoto
Prasit Palittapongarnpim
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University of Alberta
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University of Alberta
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Publication date
Application filed by University of Alberta filed Critical University of Alberta
Priority to AU58771/94A priority Critical patent/AU5877194A/en
Publication of WO1994017203A1 publication Critical patent/WO1994017203A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to a new method for detecting genomic variation. It is generally applicable to any genome or any DNA sequence which has multiple copies of an insertion sequence or other repetitive sequence.
  • the invention provides a new ligation-mediated method for PCR amplification of the DNA sequences flanking an insertion sequence.
  • the invention further provides a new rapid method for preparing DNA fingerprints which permit detection of genomic variation and provide evidence of relatedness or unrelatedness between individuals or strains within a species.
  • genomic variation within a species has proved to be a powerful tool in areas as diverse as forensic science and epidemiology and microbiology.
  • RFLP restriction fragment length polymorphism
  • RFLP has been applied to epidemiological studies of many microorganisms. For example, strain identification of M. tuberculosis has been performed by Southern blots of restriction digested chromosomal DNA with IS986 or IS6110 to detect RFLPs (Hermans, P. .M. et al; (1990), J. Clin. Microbiol. , vol. 28, pp. 2051-2058; Masurek, G.H. et al; (1991), J. Clin. Microbiol., vol. 29, pp. 2030-2033).
  • RFLP methods are, however, very laborious with an organism such as M. tuberculosis, in which purification of the required amount of DNA is notoriously difficult and such methods are not suitable for clinical diagnosis.
  • strain identification is important both epide iologically, and also clinically, for identification of particular drug resistant strains.
  • a method for identifying an individual member or a strain of a species of organism having within its DNA at least one repeated nucleotide sequence, each repeat of the repeated nucleoside sequence having a first upstream flanking sequence and a second downstream flanking sequence and the flanking sequences being polymorphic.
  • the method comprises (i) amplifying at least said first flanking sequences or said second flanking sequences by ligation-mediated polymerase chain reaction and (ii) differentiating the amplified flanking sequences on the basis of their relative size so as to characterise an individual member or strain of the species.
  • an isolated DNA molecule having the formula 5'OH-CCTTTCCAAGAACTGGAGTC-OH
  • an isolated DNA molecule having the formula
  • an isolated DNA molecule having the formula
  • a method for amplifying a portion of a DNA fragment having a known nucleotide sequence from the known nucleotide sequence to an unknown nucleotide sequence comprising (a) subjecting the DNA fragment to digestion by a restriction endonuclease to give restriction fragments having cohesive ends;
  • Figure l shows in schematic form the amplification technique employed in one embodiment of the invention.
  • Panel a shows the fate of a Bcrl II restriction fragment containing an insertion sequence (IS 6110) during the amplification process.
  • the insertion sequence IS6110 is represented by the dark grey area and the linker is represented in light grey.
  • the primers are represented by horizontal arrows.
  • Panel b shows the fate of a Bql II restriction fragment which does not contain an IS when the method is applied.
  • Panel c shows the sequence of one linker fragment of the invention (5 1 strand: Sequence ID No: 1; 3 • strand Sequence ID No: 2) and of primers dku 53 (Sequence ID No: 3) and dku 55
  • Figure 2 shows DNA fingerprinting patterns obtained by the method of the invention.
  • Lane 1 - molecular weight marker (replicative form of ⁇ xl74 digested with Hae III) .
  • Figure 3 shows RFLP patterns obtained from the samples of Figure 2.
  • Hybridising probe was IS 6110.
  • the two left hand lanes were reference strains H37Ra and
  • Lanes marked 7, 13, 14, 16, 17, 18, 15 and 27 were the clinical isolates of Figure 2, lanes 4, 5, 6, 7, 8, 9, 10 and 11.
  • Figure 4 shows DNA fingerprinting patterns obtained by the method of the invention with varying amounts of DNA.
  • Figure 5 shows DNA fingerprints obtained from organisms cultured for different time periods.
  • the inventors have devised a new method for detecting genomic variation and identifying individual members or strains within a species comprising amplification of the polymorphic sequences flanking a repeated or insertion sequences by ligation-mediation PCR followed by differentiation of the amplified flanking sequences on the basis of their relative size.
  • Amplification of the flanking DNA sequences adjacent to insertion sequences (IS) has to be achieved without knowledge of their nucleotide sequences.
  • LMPCR Blunt end ligation-mediated polymerase chain reaction
  • blunt end ligation is low efficiency and is not likely to be satisfactory as the basis of production of reproducible patterns for strain discrimination.
  • a blunt end ligation-based method is subject to artefacts if applied to DNA which has been subject to shearing during isolation, as is the case, for example, in isolation of DNA from an organism such as M. tuberculosis which has tough cell walls.
  • a new cohesive end ligation-mediated PCR method for amplifying a DNA fragment between a known nucleotide sequence and an unknown nucleotide sequence.
  • the method of the invention will be described with reference to a preferred embodiment comprising a method for identifying individual members or strains of the species M. tuberculosis.
  • the method of the invention is equally applicable to detection of genomic variation, identification of individual members or strains of, other species, including other species of mycobacteria and species of viruses, bacteria, fungi, algae, plants and animals, provided there is a repeated sequence within its DNA.
  • a method for amplifying the flanking sequences on each side of a selected IS, IS
  • the primers are complementary to the 5' terminal sequences of the two strands of IS 6110.
  • Primer dku55 is identical in sequence to the shorter strand of the linker.
  • the linker priming site is identical to one of the two IS priming sites, this strategy allows the method of the invention to be practised using two primers, rather than three primers as required by previously described methods.
  • the linker need not have the same sequence as one of the insertion sequence primers.
  • the insertion sequence primers may be complementary to any selected portions of the IS and need not be complementary to the terminal portions.
  • any repeated sequence within the M. tuberculosis genome could be used as the known sequence priming site for flanking sequence amplification.
  • linker sequence should be selected to be a sequence not occurring more frequently than in about one or two copies in the genome being analysed.
  • the linker should have cohesive ends and preferably the ligated restriction fragment should not contain a site attacked by the restriction enzyme employed.
  • the linker was, however, devised without phosphorylation, so that the longer linker strand did not become ligated to the genomic DNA and therefore did not act as a PCR priming site for the dku55 primer.
  • amplification of only one flanking sequence adjacent to an IS may also be employed, by two using primers, one complementary to the linker priming site and one complementary to the insertion sequence.
  • the amplified flanking sequences were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. Any suitable visualisation method may be employed, including primer labelling with colorimetric or radiometric detection.
  • the amplified flanking sequences may be separated on the basis of relative size in a DNA sequencer such as a Model 362 gene scanner (Applied Biosystems Inc.), with detection of a fluorescent tag on the primer giving an array of peaks which can be converted into a suitable visual signal.
  • a DNA sequencer such as a Model 362 gene scanner (Applied Biosystems Inc.)
  • detection of a fluorescent tag on the primer giving an array of peaks which can be converted into a suitable visual signal.
  • RFLP patterns of one hundred clinical isolates already examined by the method of the invention were examined by Southern blotting and probing with a BamHI-Sall fragment of pDC 73, which contains a part of the IS 6110 sequence (Masurek supra) .
  • Figure 3 shows the RFLP banding patterns of the isolates examined in Figure 2. Lanes 15 and 27 of Figure 3, the isolates from baby and mother, again shown identical banding patterns.
  • RFLP analysis of M. tuberculosis takes 48 to 72 hours, whereas the method the invention requires less than 24 hours.
  • the method of the invention is reproducible over a wide range of DNA concentrations, as seen in Figure 4 which shows banding patterns with from 0.06 to 4 nanograms of DAN.
  • RFLP requires l ⁇ g of DNA.
  • the improved speed and sensitivity of the method of the invention offer great advantages, particularly in a clinical setting.
  • Example 4 A further indication of the sensitivity of the method of the invention is provided by Example 4. Eight clinical isolates of M. tuberculosis were cultured and DNA was extracted, amplified by the method of the invention. Replicates of the same eight isolates were grown to a growth index of 100 or less and similarly treated. The paired DNA samples were examined on agarose gel, as seen in Figure 5.
  • the short term cultures contain approximately 10,000 organisms per ml, which approximates the concentration of organisms which would be expected in a smear positive sputum sample. This indicates that the sensitivity of the method of the invention is sufficient to permit application of the method directly to sputum or tissue samples, or extracts of these, without prior culturing of the organisms.
  • Sputum samples were cultured by conventional techniques to a growth index greater than 999 (Morgan et al., (1983), J. Clin. Microbiol., v. 18, pp. 384-388).
  • DNA was extracted by the following method. Mycobacteria were transferred into polypropylene tubes with siliconized glass beads and vortexed vigorously for 6-10 minutes. 600 ⁇ l of TE buffer was added and mixed. 1 ml of phenol was added for both killing the bacteria and purification of the DNA. Centrifuged for 1 minute at 3000 rp . Supernatant was transferred to a microfuge tube and centrifuged again for 10 minutes at 13000 rpm. Aqueous layer transferred to a new microfuge tube and equal volume of phenol added. Mixed well and centrifuged again for 6 minutes at 10000 rpm.
  • Aqueous phase was re-extracted 3-5 times with 1:1 phenol-chloroform mixture. After the interphase was clear, extracted once with chloroform. Two volumes of -20°C absolute ethanol were added and mixture stored in freezer for at least 30 min. Centrifuged for 10 minutes at 13000 rpm. Supernatant was drained off and pellet dried in the Speed Vac or on the bench.
  • DNA samples were diluted to a concentration of 20 ng/ ⁇ l and 100 ng DNA was digested with 20 units of BamHI per ⁇ g of DNA in M buffer (Boehringer Mannheim) for one hour.
  • a mixture containing 5 ⁇ l of the digested DNA and 10 ⁇ l of a 50 pg/ ⁇ l solution of the linker of Figure lc was warmed to 45°C for 5 min, then chilled in an ice bath.
  • Amplification was performed in a total volume of 50 ⁇ l containing 2.5 units of Tag polymerase, 0.5 ⁇ l of the ligated DNA mixture, 5 ⁇ l of lOxbuffer (100 mM Tris HCl pH 9.0, 12.5 mM MgCl 2 , 500 mM KC1, 1% triton X-100, 0.1% w/v gelatin), 200 ⁇ M of each dNTP and 0.5 ⁇ M of each of primers dku53 and dku55 (Fig lc) .
  • the reaction mix was overlaid with 50 ⁇ l of mineral oil, incubated for 3 minutes at 94°C, and subjected to 30 cycles of PCR (1 min. at 94°C, 1 min.
  • the amplified DNA fragments usually consisted of 5 to 12 bands, ranging from about 100 bp to 2 kb long.
  • a stained gel with typical DNA fingerprinting patterns is shown in Figure 2.
  • DNA from one clinical isolate was diluted to various concentrations and the method of Example 1 was carried out on 0.06, 0.12, 0.25, 0.5, 1.0, 2.0 and 4.0 ng DNA.
  • Example 3 Eight clinical isolates were divided into two portions. One portion of each was cultured to a growth index greater than 999, as described in Example 1. The other portion was cultured by the BACTEC method (Morgan, supra) , to a growth index of 100 or less. DNA was extracted from both sets of samples and processed as in Example 1.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

Procédé permettant d'identifier un élément individuel ou une souche d'une espèce d'un organisme, comprenant l'amplification d'au moins une séquence adjacente à une séquence répétée dans l'ADN de l'élément ou de la souche et la différenciation des séquences adjacentes amplifiées fondée sur leur taille relative afin de caractériser un élément individuel ou une souche de l'espèce.
PCT/CA1994/000033 1993-01-29 1994-01-24 Procede d'analyse d'empreinte d'adn amplifie permettant de detecter les variations du genome Ceased WO1994017203A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU58771/94A AU5877194A (en) 1993-01-29 1994-01-24 Amplified dna fingerprinting method for detecting genomic variation

Applications Claiming Priority (2)

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GB9301776.2 1993-01-29
GB939301776A GB9301776D0 (en) 1993-01-29 1993-01-29 Method for detecting genomic variation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0721987A1 (fr) * 1995-01-16 1996-07-17 Keygene N.V. Amplification de séquences répétitives simples
WO1996017082A3 (fr) * 1994-11-28 1996-08-08 Du Pont Sondes de microsatellites composes pour la detection de polymorphismes genetiques
WO1997016564A1 (fr) * 1995-11-01 1997-05-09 Chiron Diagnostics Corp. Detection moleculaire amelioree du mycobacterium tuberculosis faisant intervenir l'element d'insertion is6110
US6218119B1 (en) 1996-01-16 2001-04-17 Keygene, N. V. Amplification of simple sequence repeats

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0461045A1 (fr) * 1990-06-08 1991-12-11 Institut Pasteur Détection spécifique du mycobacterium tuberculosis
WO1992013104A1 (fr) * 1991-01-22 1992-08-06 Board Of Regents, The University Of Texas System Progression d'une reaction en chaine de polymerase 5' et 3' a partir de sequences d'adn connues
EP0534858A1 (fr) * 1991-09-24 1993-03-31 Keygene N.V. Amplification sélective des fragments de restriction: procédé général pour le "fingerprinting" d'ADN
WO1993008297A1 (fr) * 1991-10-23 1993-04-29 Baylor College Of Medicine Determination d'empreintes relatives a des souches bacteriennes par amplification repetitive de sequences d'adn

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0461045A1 (fr) * 1990-06-08 1991-12-11 Institut Pasteur Détection spécifique du mycobacterium tuberculosis
WO1992013104A1 (fr) * 1991-01-22 1992-08-06 Board Of Regents, The University Of Texas System Progression d'une reaction en chaine de polymerase 5' et 3' a partir de sequences d'adn connues
EP0534858A1 (fr) * 1991-09-24 1993-03-31 Keygene N.V. Amplification sélective des fragments de restriction: procédé général pour le "fingerprinting" d'ADN
WO1993008297A1 (fr) * 1991-10-23 1993-04-29 Baylor College Of Medicine Determination d'empreintes relatives a des souches bacteriennes par amplification repetitive de sequences d'adn

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
A. ROSENTHAL AND D.S.C. JONES: "Genomic walking and sequencing by oligo-cassete mediated polymerase chain reaction", NUCL. ACIDS RES., vol. 18, no. 10, 25 May 1990 (1990-05-25), IRL PRESS, OXFORD, ENGLAND;, pages 3095 - 3096 *
D. KUNIMOTO ET AL.: "DNA fingerprinting of Mycobacterium tubercolosis isolates by ligation mediated polymerase chain reaction", ABSTR. GEN. MEET. AM. SOC. MICROBIOL., vol. 93, no. 0, 1993, pages 169 *
J.P. KIM ET AL.: "Sequence independent single primer amplification (SISPA): Utility and application to the detection of enterically transmitted non-A, non-B hepatitis (ET-NANBH)", J. CELL. BIOCHEM. (SUPPL. 13 PART E), 3 April 1989 (1989-04-03), pages 293 *
K.D. EISENACH ET AL.: "Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tubercolosis", JOURNAL OF INFECTIOUS DISEASES, vol. 161, no. 5, May 1990 (1990-05-01), THE UNIVERSITY OF CHICAGO, US;, pages 977 - 981 *
P. PALITTAPONGARNPIM ET AL.: "DNA fingerprinting of Mycobacterium tubercolosis isolates by ligation-mediated polymerase chain reaction", NUCL. ACIDS RES., vol. 21, no. 3, 11 February 1993 (1993-02-11), IRL PRESS, OXFORD, ENGLAND;, pages 761 - 762 *
P. PALITTAPONGARNPIM ET AL.: "Tuberculosis contact follow-up directed by arbitrary polymerase chain reaction DNA fingerprinting of M. tuberculosis", PROGRAM ABSTR. INTERSCI. CONF. ANTIMICROB. AGENTS CHEMOTHERAPY, vol. 32, no. 0, 1992, pages 203 *
P.W.M. HERMANS ET AL.: "Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tubercolosis complex strains", INFECTION AND IMMUNITY, vol. 59, no. 8, August 1991 (1991-08-01), AM. SOC. MICROBIOL., BALTIMORE, US;, pages 2695 - 2705 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017082A3 (fr) * 1994-11-28 1996-08-08 Du Pont Sondes de microsatellites composes pour la detection de polymorphismes genetiques
EP0721987A1 (fr) * 1995-01-16 1996-07-17 Keygene N.V. Amplification de séquences répétitives simples
WO1996022388A1 (fr) * 1995-01-16 1996-07-25 Keygene N.V. Amplification de sequences repetitives simples
US5874215A (en) * 1995-01-16 1999-02-23 Keygene N.V. Amplification of simple sequence repeats
WO1997016564A1 (fr) * 1995-11-01 1997-05-09 Chiron Diagnostics Corp. Detection moleculaire amelioree du mycobacterium tuberculosis faisant intervenir l'element d'insertion is6110
US5731150A (en) * 1995-11-01 1998-03-24 Chiron Diagnostic Corporation IS6110 based molecular detection of mycobacterium tuberculosis
US6218119B1 (en) 1996-01-16 2001-04-17 Keygene, N. V. Amplification of simple sequence repeats

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GB9301776D0 (en) 1993-03-17

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