WO1995023236A1 - Methode d'utilisation de sites d'amorcage mobiles pour le sequençage de l'adn - Google Patents

Methode d'utilisation de sites d'amorcage mobiles pour le sequençage de l'adn Download PDF

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Publication number
WO1995023236A1
WO1995023236A1 PCT/US1995/000503 US9500503W WO9523236A1 WO 1995023236 A1 WO1995023236 A1 WO 1995023236A1 US 9500503 W US9500503 W US 9500503W WO 9523236 A1 WO9523236 A1 WO 9523236A1
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WIPO (PCT)
Prior art keywords
dna
segment
sequencing
restriction
prinker
Prior art date
Application number
PCT/US1995/000503
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English (en)
Inventor
Jack T. Leonard
Original Assignee
Amicon, 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 Amicon, Inc. filed Critical Amicon, Inc.
Priority to AU15668/95A priority Critical patent/AU1566895A/en
Publication of WO1995023236A1 publication Critical patent/WO1995023236A1/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
    • C12Q1/6869—Methods for sequencing

Definitions

  • This invention relates to improvements in DNA characterization involving the use of mobile DNA priming sites for DNA sequencing.
  • the desired end-product of most DNA characterization procedures is the nucleotide sequence of the DNA.
  • the laboratory procedures which commonly precede DNA sequencing are 1) restriction mapping, and 2) subcloning or polymerase chain reaction ("PCR") amplification.
  • the function of subcloning is to divide large DNA fragments into smaller fragments which can be propagated in a suitable host, thereby providing large amounts of template (the DNA segments or fragments of interest that are being cloned) for sequencing.
  • the subcloning procedure also provides primer sites carried on the vector DNA which can be used in DNA sequencing.
  • DNA sequencing and PCR rely on the ability of a DNA polymerase to extend an oligonucleotide primer that is annealed to a single stranded DNA template.
  • a thermostable DNA polymerase is used with a single oligonucleotide primer.
  • PCR ordinarily extends primers from two complementary DNA strands, and thus makes double stranded DNA product, whereas asymmetric PCR produces predominantly single-stranded DNA by limiting the concentration of one primer while adding an excess of another.
  • the primer must be complementary to the primer site on the template DNA, i.e., the primer site on the template DNA and the oligonucleotide primer must be closely matched to ensure that annealing takes place at one location. (There must of course also be a primer sequence on the template DNA.) Since the primer sites are known on vector DNA (and on the ends of PCR product), these can be sequenced directly; however, due to practical limitations of sequencing technology, DNA may only be sequenced from the priming site to a maximum length of about 300 nucleotides (“nt"). If the DNA sequence on the ends of restriction fragments were known, complementary primers could be synthesized, and the DNA sequenced.
  • the invention relates to a method of sequencing DNA, comprising the steps of a) identifying on a DNA molecule a segment containing genetic information of interest; b) selecting a restriction enzyme that will excise the segment and leave at least one end of the segment with a sticky end; c) making a restriction digest by treating the DNA molecule with the restriction enzyme, excising the segment and leaving at least one end of the segment with a sticky end; d) selecting a prinker comprising a double- stranded oligonucleotide portion having a defined nucleotide sequence that is recognized as a "starting sequence" for a DNA polymerase, and at least one single- stranded sticky end with a sequence of nucleotides complementary to the sticky end of the segment; e) ligating the prinker to the segment to form an activated DNA segment; f) isolating the activated DNA segments from the restriction digest; and g) sequencing the activated DNA segments.
  • the invention describes a method by which "sticky ended" restriction fragments are ligated to double stranded DNA oligonucleotides of known sequence (i.e., linkers) having a sticky end complementary to that on the restriction fragments, with the resulting "activated" DNA molecules isolated and subjected to DNA sequencing.
  • linkers One strand of the linker serves as the priming site for DNA sequencing. It is proposed that this class of specialized oligonucleotides be termed "prinkers" to indicate the combination of priming and linking functions.
  • Primer is used herein in accordance with its generally accepted definition: (single-stranded) oligonucleotides which will anneal to complementary priming sites by virtue of specific hydrogen- bonding interactions, and, in particular, oligonucleotides which are used to anneal to target sequences, i.e., DNA polymerase priming sites.
  • Linker is also used herein in accordance with its generally accepted definition: double-stranded DNA "adapters” which are ligated onto the end of restriction fragments. After a prinker has been ligated to a target DNA segment containing genetic information of interest, the segment is now what may be called, for present purposes, "activated” and may be easily sequenced.
  • the activated DNA segment can be sequenced directly once it has been isolated from the rest of the restriction digest (e.g., by gel electrophoresis). If there is not a sufficient amount of the activated DNA segment available for sequencing the segment may alternately be amplified by thermal cycle sequencing ("TCS"), PCR, or other technique that increases the number of copies of the activated DNA segment. This may be done by amplifying the whole digest to increase the amount of the segment of interest.
  • TCS thermal cycle sequencing
  • a library of prinkers having sticky ends complementary to the characteristic nucleotide overhang sequences of all known endonucleases is within the scope of the invention.
  • a kit for sequencing DNA in accordance with the invention may be obtained.
  • DNA sequencing in accordance with the invention of any desired sequence may be accomplished because no matter what restriction enzyme is used in the restriction digest, there will be a prinker in the kit that may be ligated to the fragment(s) of interest.
  • Type II endonucleases produce specific single stranded overhangs, either two or four nucleotides in length. These overhangs, known as “sticky ends", can be ligated to the complementary ends of another DNA molecule, using T4 DNA ligase. Different DNA fragments that can be ligated together by virtue of their sticky ends are said to possess compatible cohesive ends. Other endonucleases either generate "blunt ends” or a variety of sticky ends. These would be of limited use because blunt ends ligate poorly, and unknown sticky ends can not be specifically targeted for ligation. As such, sticky ends corresponding to Type II endonuclease sequences, shown in Table I, are preferred in the invention. TABLE I.
  • the use of the invention would obviate the problems associated with maintenance and propagation of DNA in organisms and allow for the generation of large amounts of virtually any restriction fragment, by attaching a prinker, having a defined primer sequence, to the ends of a restriction fragment, which could then be amplified if necessary, before isolation and sequencing.
  • the resultant product(s) could be isolated and restricted again. This process could be repeated indefinitely, circumventing subcloning procedures altogether.
  • the identity of restriction sites on the ends of restriction fragments from multiple restriction digests could be identified using different combinations of prinkers, followed by ligation and amplification. Restriction sites encoded on the prinkers could facilitate their cloning if desired.
  • a prinker containing the priming site specific for that enzyme may be used in the procedure.
  • prinkers having the priming site for that enzyme may be used.
  • Another advantage is that it is easier and more efficient to incorporate radioactive nucleotides; because the primer nucleotide sequence is known, one knows exactly which "hot" nucleotides to incorporate into the polymerase mixture.
  • Another embodiment of the invention may be found where the restriction fragments generated by an endonuclease are sufficiently abundant to sequence without amplification.
  • the alkaline phosphatase removes the 5'-terminal phosphate from the restriction fragment; thus, when the prinker is attached to the restriction fragment, only one of the two complementary oligonucleotide strands (the phosphorylated strand) of the prinker is ligated to the restriction fragment.
  • the ligated oligonucleotide then serves as a "naked" priming site during DNA sequencing.
  • the priming site is "naked" in the sense that ordinarily the primer must compete with one of the two strands of the double stranded DNA template for the priming site, which necessitates denaturation steps. Since the top strand of the prinker, which occupies the priming site, is not covalently attached, to the restriction fragment, it can serve as the primer and can be easily displaced.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
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  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode de séquençage de gènes d'ADN, faisant appel à des sites mobiles d'ADN pour les ADN-polymérases utilisées pour le séquençage de l'ADN.
PCT/US1995/000503 1994-02-25 1995-01-11 Methode d'utilisation de sites d'amorcage mobiles pour le sequençage de l'adn WO1995023236A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15668/95A AU1566895A (en) 1994-02-25 1995-01-11 Method of using mobile priming sites for dna sequencing

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US20240094A 1994-02-25 1994-02-25
US08/202,400 1994-02-25

Publications (1)

Publication Number Publication Date
WO1995023236A1 true WO1995023236A1 (fr) 1995-08-31

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PCT/US1995/000503 WO1995023236A1 (fr) 1994-02-25 1995-01-11 Methode d'utilisation de sites d'amorcage mobiles pour le sequençage de l'adn

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AU (1) AU1566895A (fr)
WO (1) WO1995023236A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002673A1 (fr) * 1994-07-14 1996-02-01 Amicon, Inc. PROCEDE D'UTILISATION DE SITES D'AMORÇAGE MOBILES POUR LE SEQUENçAGE DE L'ADN

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001582A1 (fr) * 1992-07-13 1994-01-20 Medical Research Council Procede de categorisation de populations de sequences nucleotidiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001582A1 (fr) * 1992-07-13 1994-01-20 Medical Research Council Procede de categorisation de populations de sequences nucleotidiques

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AKANE, A. ET AL.: "direct dideoxy sequencing of genomic DNA by ligation mediation", BIOTECHNIQUES, vol. 16, no. 2, US, pages 238 - 41 *
COLLASIUS, M. ET AL.: "analysis of unknown DNA sequences by polymerase chain reaction (PCR) using a single specific primer and a standardized adaptor", JOURNAL OF VIROLOGICAL METHODS, vol. 32, NL, pages 115 - 19 *
HETZER, M. ET AL.: "PCR mediated analysis of RNA sequences", NUCLEIC ACIDS RESEARCH, vol. 21, no. 3, GB, pages 5526 - 27 *
KALISCH, B. ET AL: "covalent linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments", GENE, vol. 44, NL, pages 263 - 270 *
PALITTAPONGARNPIM, P. ET AL.: "DNA fingerprinting of mycobacterium tuberculosis isolates by ligation-mediated polymerase chain reaction", NUCLEIC ACIDS RESEARCH, vol. 21, no. 3, GB, pages 761 - 62 *
ROSENTHAL, A. ET AL.: "genomic walking and sequencing by cassette mediated polymerase chain reaction", NUCLEIC ACIDS RESEARCH, vol. 18, GB, pages 3095 - 96 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002673A1 (fr) * 1994-07-14 1996-02-01 Amicon, Inc. PROCEDE D'UTILISATION DE SITES D'AMORÇAGE MOBILES POUR LE SEQUENçAGE DE L'ADN

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Publication number Publication date
AU1566895A (en) 1995-09-11

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