WO2002074994A2 - Methode de sequençage orestes amelioree - Google Patents

Methode de sequençage orestes amelioree Download PDF

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Publication number
WO2002074994A2
WO2002074994A2 PCT/US2001/046665 US0146665W WO02074994A2 WO 2002074994 A2 WO2002074994 A2 WO 2002074994A2 US 0146665 W US0146665 W US 0146665W WO 02074994 A2 WO02074994 A2 WO 02074994A2
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Prior art keywords
cell
nucleic acid
organism
sequences
cdna
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PCT/US2001/046665
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English (en)
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WO2002074994A3 (fr
Inventor
Andrew John George Simpson
Emmanuel Dias-Neto
Ricardo R. Brentani
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Ludwig Institute For Cancer Research
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Publication of WO2002074994A2 publication Critical patent/WO2002074994A2/fr
Publication of WO2002074994A3 publication Critical patent/WO2002074994A3/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • 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

  • the single stranded cDNA is prepared, it is used in an amplification reaction.
  • the single primer used is identical to the first primer, as described supra, and that low stringency conditions be employed. Using identical primers tends to produce longer products, but this is not required.
  • the darker portion is a sequence obtained in accordance with the invention.
  • RNA samples from S. mansoni DNA and human cancer tissues were collected from freshly perfused hamsters, and frozed immediately.
  • Total RNA was isolated from approximately 500 mg of tissue, using standard methods.
  • Messenger RNA was isolated using a commercially available mRNA kit, resuspended in 40 ⁇ of buffer, and then treated with Dnase (lU/10 ⁇ ), for 15 minutes, at room temperature. The DNase was inactivated by incubating at 65 °C for 10 minutes. Integrity of mRNA was checked routinely by performing control amplification of three different messages using 3 different pairs of primers which match the 5' end of S. mansoni control genes. The control genes were actin (abundant message), retinoic X receptor, 4211 bases (long mRNA), and catepsin C (low abundance message). Controls showed that the mRNA was intact.
  • PROTOCOL 3 S. MANSOND:
  • Protocol 1 was used on 333 different ORESTES cDNA libraries which had been derived from three different human tissues, and four different mRNA preparations. The clones that were derived from this set of libraries were sequenced, yielding 65,091 ESTs, with an average of 195 ESTs per minilibrary. This sequence set was used for normalization analysis, and positional distribution. Analysis of normalization was performed using 11,848 ESTs, which matched complete human genes present in the UniGene cluster. The average size of the clusters tagged by the sequences generated was 495. Size distribution is set forth in figure 4. Positional distribution is shown in figure 5. The central portion of the transcripts is preferentially tagged.
  • Steps 2-38 are then repeated, 26 times 39) 72 °C 30 seconds PROTOCOL 5
  • a set of approximately 250,000 ESTs was generated, and then analyzed.
  • the ESTs were size selected, in distinct size ranges, varying from 0.3 to 1.5kb.
  • the size selected fragments were cloned into pUC18, using standard methods.
  • the plasmids were cloned into cells, and colonies were grown overnight in liquid media. PCR was carried out thereafter, and the PCR products were used for DNA sequencing.
  • sequences were trimmed to exclude primer sequences and vector sequences, as well as low quality regions.
  • the sequences were clustered, using "CAP3", in accordance with Huang, et al, Genome Res 9:868-877 (1999), inco ⁇ orated by reference. Preliminary analysis of the sequences showed that 18% of the sequences were derived from rRNA and mtDNA transcript, or were composed almost entirely of repetitive sequences. These were excluded from further analysis.
  • the method involves forming a cDNA library by contacting a sample of mRNA with at least one arbitrary primer, at low stringency conditions, followed by reverse transcription. The resulting, single stranded cDNA is then amplified, with at least one arbitrary primer, at low stringency, to create a mini-library of cDNA.
  • a cDNA library by contacting a sample of mRNA with at least one arbitrary primer, at low stringency conditions, followed by reverse transcription.
  • the resulting, single stranded cDNA is then amplified, with at least one arbitrary primer, at low stringency, to create a mini-library of cDNA.
  • These nucleotide sequences are derived from internal, coding regions of mRNA.
  • the resulting nucleic acid molecules are then sequenced.
  • the improvement comprises varying the annealing temperature for the amplification reaction, wherein a high annealing temperature is used, for a number of cycles, followed by a number of cycles with a low annealing temperature.
  • a source of pre-existing sequence information e.g., a nucleotide sequence library.
  • the improvement comprises varying the annealing temperature for the amplification reaction, wherein a high annealing temperature is used, for a number of cycles, followed by a number of cycles with a low annealing temperature.
  • pre-existing information which corresponds to internal mRNA sequences can be identified.
  • the method is applied to eukaryotes.
  • the method as described herein is applicable to any organism, including single cell organisms such as yeast, parasites such as Plasmodium, and multicellular organisms. All plants and animals, including humans, can be studied in accordance with the methods described herein.
  • a second feature of the invention is a method for developing so-called "contig" sequences.
  • These are nucleotide sequences which are generated following comparing sequences produced in accordance with this method to previously determined sequences, to determine if there is overlap. This is of interest because longer sequences are of great interest in that they define the target molecule with much greater accuracy.
  • These contigs may be produced by comparing sequences developed in accordance with the method, as well as by comparing the sequences to pre-existing sequences in a databank. The aim is simply to find overlap between two sequences.
  • the power of the inventive method is such that there are innumerable applications. For example, it is frequently desirable to carry out analyses of populations of subjects.
  • the invention can be used to carry out genetic analyses of large or small populations. Further, it can be used to study living systems to determine if, e.g., there have been genetic shifts which render an individual or population more or less likely to be afflicted with diseases such as cancer, to determine antibiotic resistance or non-tolerance, and so forth.
  • the invention can also be used in the study of congenital diseases, and the risk of affliction to a fetus, as well as the study of whether such conditions are likely to be passed to offspring via ova or sperm.
  • analyses for pathological conditions can be carried out in all animals, plants, birds, fish, etc.
  • the invention is applicable to all eukaryotes, not just humans, and not just animals.
  • the genomes of food crops can be studied to determine if resistance genes are present, have been inco ⁇ orated into a genome following transfection, and so forth. Defects in plant genomes can also be studied in this way.
  • the method permits the artisan to determine when pathogens which integrate into the genome, such as retroviruses and other integrating viruses, such as influenza virus, have undergone shifts or mutations, which may require different approaches to therapy.
  • This aspect of the invention can also be applied to eukaryotic pathogens, such as trypanosomes, different types of Plasmodium, and so forth.
  • the method described herein can also be applied to DNA directly. More specifically, there are organisms, such as particular types of bacteria, which are very difficult to culture. One can apply the inventions described herein to DNA of these or other bacteria directly, rather than to cDNA prepared from mRNA. Essentially, the methodology used is the same as the methodology described supra, except genomic DNA is used. In such a case, random fragments are produced, rather than ORF segments. Using PCR in this type of approach means that very small amounts of DNA are needed, hence difficulties in culture are avoided. It is estimated that less than one microgram of DNA would be necessary to sequence an entire genome of a prokaryote.
  • annealing temperature ranging from 72 °C to 30 °C, more preferably about 60 ° C to 40° C are preferred. These so-called “touchdowns” are used at least once, more preferably up to 40 or 50 times. More preferably, they are used 1-30 times. Essentially, denaturing, annealing of abundant message, annealing of primers, and then primer extension constitute one cycle, and the temperature may vary at each of the steps.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (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 destinée à obtenir des informations sur des séquences nucléotidiques à partir de molécules d'acides nucléiques, et notamment de l'ADN complémentaire. Cette méthode consiste à faire intervenir des amorces arbitraires et des conditions de faible stringence. Plutôt que de fournir des informations à partir des terminaisons des molécules d'acides nucléiques, cette méthode fournit des informations sur des parties internes plus utiles et intéressantes des molécules d'acides nucléiques. Ladite méthode montre comment sécuriser les informations sur les ORF et comment préparer des séquences de clones chevauchants à partir d'une source quelconque.
PCT/US2001/046665 2000-11-07 2001-11-01 Methode de sequençage orestes amelioree WO2002074994A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24631300P 2000-11-07 2000-11-07
US60/246,313 2000-11-07

Publications (2)

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WO2002074994A2 true WO2002074994A2 (fr) 2002-09-26
WO2002074994A3 WO2002074994A3 (fr) 2003-08-14

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487985A (en) * 1990-10-15 1996-01-30 Stratagene Arbitrarily primed polymerase chain reaction method for fingerprinting genomes
WO2000031299A2 (fr) * 1998-11-20 2000-06-02 Ludwig Institute For Cancer Research Procede de determination de sequences nucleotidiques utilisant des amorces primaires et une faible stringence
WO2001051518A2 (fr) * 2000-01-14 2001-07-19 Ludwig Institute For Cancer Research Molecules d'acide nucleique isolees codant pour une molecule de semaphorine humaine et utilisations correspondantes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487985A (en) * 1990-10-15 1996-01-30 Stratagene Arbitrarily primed polymerase chain reaction method for fingerprinting genomes
WO2000031299A2 (fr) * 1998-11-20 2000-06-02 Ludwig Institute For Cancer Research Procede de determination de sequences nucleotidiques utilisant des amorces primaires et une faible stringence
WO2001051518A2 (fr) * 2000-01-14 2001-07-19 Ludwig Institute For Cancer Research Molecules d'acide nucleique isolees codant pour une molecule de semaphorine humaine et utilisations correspondantes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIAS NETO E ET AL: "Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA" GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 186, 1997, pages 135-142, XP002138178 ISSN: 0378-1119 cited in the application *
EMMANUEL DIAS NETO ET AL: "SHOTGUN SEQUENCING OF THE HUMAN TRANSCRIPTOME WITH ORF EXPRESSED SEQUENCE TAGS" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 97, no. 7, 28 March 2000 (2000-03-28), pages 3491-3496, XP000996193 ISSN: 0027-8424 cited in the application *
RADELOFF ET AL: "PRESELECTION OF SHOTGUN CLONES BY OLIGONUCLEOTIDE FINGERPRINTING: AN EFFICIENT AND HIGH THROUGHPUT STRATEGY TO REDUCE REDUNDANCY IN LARGE-SCALE SEQUENCING PROJECTS" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 26, no. 23, December 1998 (1998-12), pages 5358-5364, XP002103597 ISSN: 0305-1048 *

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WO2002074994A3 (fr) 2003-08-14

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