WO2003014384A1 - Nouveau procede d'elimination d'une sequence de repetition dans le genome, et sa mise en oeuvre pour la preparation de puces genomiques - Google Patents

Nouveau procede d'elimination d'une sequence de repetition dans le genome, et sa mise en oeuvre pour la preparation de puces genomiques Download PDF

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Publication number
WO2003014384A1
WO2003014384A1 PCT/CN2001/001208 CN0101208W WO03014384A1 WO 2003014384 A1 WO2003014384 A1 WO 2003014384A1 CN 0101208 W CN0101208 W CN 0101208W WO 03014384 A1 WO03014384 A1 WO 03014384A1
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Prior art keywords
alu
artificial chromosome
sequence
primers
specific
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Ceased
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PCT/CN2001/001208
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English (en)
Chinese (zh)
Inventor
Xinyuan Guan
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University of Hong Kong HKU
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University of Hong Kong HKU
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Priority to CNB018236065A priority Critical patent/CN1288251C/zh
Priority to PCT/CN2001/001208 priority patent/WO2003014384A1/fr
Publication of WO2003014384A1 publication Critical patent/WO2003014384A1/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
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a novel method for eliminating genomic repetitive sequences in the preparation of genomic chips using artificial chromosomes.
  • the genomic DNA sequence obtained by this new method does not contain genomic repeat sequences, so it effectively eliminates non-specific hybridization signals caused by repeated sequences and improves the accuracy of the genomic chip. Background technique
  • Artificial chromosomes include yeast artificial chromosome (Yeast Artficial Chromosome, YAC. Its insert is about 1,000 kilobase pairs), phage artificial chromosome (Phage Artificial Chromosome, PAC. Its insert is about 100 kilobase pairs), bacteria Artificial chromosome (Bacterial Art ificial Chromosome, BAC. Its insert is about 100 kilobase pairs).
  • Such artificial chromosomes can be used as chromosome fluorescence in site hybridization (FISH) probes for a variety of purposes, including genetic disease diagnosis (mainly various chromosomal abnormalities), prenatal diagnosis (pregnant women Genetic disease screening), tumor typing, diagnosis and prognosis tests, to observe radiation and other environmental factors for human damage.
  • genetic disease diagnosis mainly various chromosomal abnormalities
  • prenatal diagnosis pregnant women Genetic disease screening
  • tumor typing diagnosis and prognosis tests
  • diagnosis and prognosis tests to observe radiation and other environmental factors for human damage.
  • FISH chromosome fluorescence in site hybridization
  • Alu sequence There are many repeated DNA sequences in the human genome. These sequences are very similar and appear consistently throughout the genome. Among them, the Alu sequence appears most frequently, appearing about every 4 kilobase pairs. The Alu sequence accounts for about 5% of the total human genome DNA, up to 900,000 copies, of which 60% of the sequence members contain the cut of the restriction enzyme Alu, so it is called the Alu family, which is mainly concentrated in the R band in the late stage of cell division. It belongs to non-coding DNA, but some of it is located in the untranslated region of mRNA, even in the coding region. The longest Alu sequence is about 150 base pairs. Human Alu
  • the sequence is about 300bp in length, and is composed of 120bp and 150bp repeats. The two are separated by A-rich regions, and there is a 7-10bp forward repeat at both ends. Alu sequences are not exactly the same, there are some differences. These repeats can interfere with specific hybridization signals because they can hybridize to each other to generate non-specific background signals. This is currently the biggest challenge faced by using artificial chromosomes to make probes or make genomic chips.
  • the purpose of the present invention is to provide a new method for amplifying human genomic DNA.
  • the method can effectively remove the repeated sequences when amplifying the human genomic DNA sequence in an artificial chromosome, thereby eliminating non-specific hybridization caused by the repeated sequences Signal interference, improve the accuracy and reliability of hybridization results.
  • a polymerase chain reaction amplification method in which an artificial chromosome or a large fragment DNA molecule having a length of 50-5000 kb is used as a template, and an Alu-specific primer is used as a primer, wherein Alu-specific primers specifically bind to the 5 'end of the Alu sequence and extend in the 3'-5' direction of the Alu sequence, or both bind to the 3 'end of the Alu sequence and extend in the 5' direction of the Alu sequence — 3 'direction.
  • the length of the Alu-specific primer is 15-25 bp.
  • the Alu-specific primer is selected from the following group:
  • a method for generating a gene chip comprises the steps of: (a) amplifying a polynucleotide product by a polymerase chain reaction, in which an artificial chromosome or A large DNA molecule with a length of 50-5000kb is used as a template, and Alu-specific primers are used as primers.
  • the Alu-specific primers specifically bind to the 5 'end of the Alu sequence and extend in the 3' direction of the Alu sequence ⁇ 5 'direction, or both bound to the 3' end of the Alu sequence and extending in the 5 ' ⁇ 3' direction of the Alu sequence; (b) Spotting the polynucleotide product in step (a) on the gene chip substrate to form a gene chip.
  • a gene (group) chip prepared by the above method is also provided.
  • Figure 1 is an electrophoresis image of the PCR product amplified with Alu primers, where the template is the DNA of the BAC clone.
  • the molecular weight marker (M) is a 1 kb DNA ladder.
  • RPII-497 I 24 etc. are the numbers of BAC clones, where RPII is the name of the BAC clone library (consisting of many plates), and the set of numbers after "RP11-" (such as 497) is the name of one of the plates in the library
  • the following English letters (such as I) and another set of numbers (such as 24) refer to the abscissa and ordinate of the position of the BAC clone in this disc, respectively.
  • Figure 2 is a Southern blot of PCR products amplified with Alu primers.
  • the molecular weight marker (M) is a 1 kb DNA ladder.
  • Positive controls 1 and 2 are BAC DNA.
  • the entire BAC clone DNA is used as a probe.
  • the BAC clone is numbered RPII-110-0-7 and has a size of 120kb. detailed description
  • artificial chromosome refers to an artificially constructed, self-replicating vector with chromosomal characteristics (such as containing centromere, telomere, and DNA replication origin, but also with a transformation screening marker).
  • Representative artificial chromosomes include, but are not limited to: Yeast Artificial Chromosome (YAC), Phage Artificial Chromosome (PAC), Bacterial Artificial Chromosome (BAC), Mammalian Artificial Chromosome (MAC).
  • YAC uses yeast chromosomes centromeres, telomeres, and so on.
  • Alu-specific primer refers to a primer that specifically binds to the end of an Alu sequence such that the polymerase chain reaction amplification product contains no or substantially no Alu repeats.
  • Alu-specific primers are divided into two types: The first type of Alu-specific primers specifically bind to the 5 'end of the Alu sequence and extend in the 3' ⁇ 5 'direction of the Alu sequence; the second type of Alu-specific primers are Binding to the 3 'end of the Alu sequence and the extension direction is the 5'-3' direction of the Alu sequence.
  • This method uses uniquely designed amplification primers and amplification conditions, where the amplification primers are located at the 5 'and 3' ends of the Alu repeats that occur at high frequency in human genomic DNA.
  • the characteristic of this method is that by using this primer to amplify the human genomic DNA sequence in, for example, bacterial artificial chromosome (BAC) by PCR method, it is possible to effectively remove the high-frequency Alu repeats appearing in human genomic DNA.
  • BAC bacterial artificial chromosome
  • the principle of this technical method is to take advantage of the frequency of Alu sequences, and use Alu-end sequences as templates to design corresponding primers.
  • the two Alu sequences are not far apart, that is, within a normal PCR reaction amplification range (generally within 2 kilobase pairs) and the directions of the two Alu sequences are opposite (such as 5 'end to 5' end)
  • the genomic DNA sequence between these two Alu sequences can be amplified. Because the DNA sequence amplified by this method is located between two Alu repeats, it no longer contains repeats.
  • BAC bacterial artificial chromosomes
  • their size is generally around 100 kilobase pairs.
  • Alu sequences once every 4 kilobase pairs, usually there can be about 25 Alu sequences in a BAC, and a considerable part of them are separated by the PCR reaction amplification range. Therefore, when using the Alu-specific primers of the present invention to amplify human genomic DNA in BAC, the chance of obtaining a single sequence can be greatly increased.
  • the PCR reaction conditions are not particularly limited, and conventional specific amplification PCR conditions can be used in the present invention.
  • a common condition is denaturation at 90-95 ° C for 45-75 seconds, annealing at 50-65 ° C for 30-90 seconds, and 70-74 ⁇ extension for 30-90 seconds, for a total of 25-35 cycles.
  • the experimental results prove the feasibility and high efficiency of this new technical method.
  • the inventors selected 100 BAC clones from human chromosome 3 and 16 and amplified them with specific primers at the 5 'and 3' ends of the Alu sequence designed by the present invention, respectively. All BACs have at least one PCR product, and up to a dozen PCR products.
  • a BAC containing multiple repeats was used as a probe (RP-11-110-0-7). After the radioisotope is labeled with 32 P, a molecular hybridization reaction is performed with these PCR products. The results showed that all the PCR products contained no repeats.
  • DNA sequencing was performed on some PCR products. The sequencing results also proved that the size of these PCR products was between 400-2,000 base pairs, with an average of about 800 base pairs, and they did not contain repeats.
  • a single human genomic DNA sequence without repeating sequences can be rapidly amplified from artificial chromosomes or large DNA fragments such as BAC clones.
  • the technology of the present invention has a variety of uses, including but not limited to-(1). These amplified single sequences can be used as genetic probes for Southern blot molecular hybridization;
  • the oligonucleotide or a fragment thereof obtained by the method of the present invention can be used as a subject of a microarray.
  • the microarray column can be used to detect the expression level of a large number of genes at the same time, and can detect gene mutations, mutations and polymorphisms. This information can be used to infer gene function, understand the genetic basis of disease, and diagnose diseases.
  • Microarray is also called DNA chip. This refers to the arrangement of different polynucleotides on a substrate, such as paper, nylon or any other membrane, filter, chip, glass slide or any other suitable solid support.
  • element or “microarray element” refers to a hybridizable polynucleotide arranged on the surface of a substrate.
  • the DNA chip can be obtained by using a series of chemical methods and inkjet technology to fix the sample on the substrate. Commonly used methods include radiation, chemistry, thermodynamics, mechanical methods, drying, etc. The formed elements are shaped like dots, bars, etc. A typical chip usually contains a certain number of components, which can be prepared by hand or with appropriate equipment. After the hybridization reaction, the unbound probe is washed away, and then a scanner is used to detect the components and the degree of the reaction that occurred. The complementarity and binding amount of the probe to each chip element can be judged by analyzing the scanned image. A common method is to directly sequence the PCR products amplified by Alu-specific primers on a glass or other carrier, and then pass It is fixed by UV cross-linking and drying.
  • Bacterial artificial chromosome (BAC) clones are cultured, coated on a culture plate, cultured, single clones are picked, the cloned bacteria are shaken, and BAC DNA is extracted from the bacteria.
  • BAC Bacterial artificial chromosome
  • the method for amplifying BAC DNA fragments without repeats by PCR is as follows: Take 50 ng BAC DNA (as a template for PCR reaction), and perform the reaction in a 50 ⁇ 1 (microliter) reaction volume.
  • the components in this reaction system are: 10 mM Tris-HCl, pH 8. 4, 2 mM MgCl, 50 mM KCl, 0.1 mM gelatin, 200 raM Dntp, 0.5 mM primers Alu- Nl or Alu- N2, 2 units Taq polymerase).
  • the conditions for the PCR reaction were: 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C. C for 2 minutes, 30 cycles.
  • Example 2 The PCR product obtained in Example 2 was subjected to gel electrophoresis and then transferred to a nylon membrane. 32 P-labeled intact BAC clone (RP11-110-0-7) DNA was used as a probe to hybridize to the Southern blot membrane.
  • the PCR amplification product obtained above was cloned into a TA-Vector vector for storage and subsequent replication in large quantities.
  • the insert was amplified by a PCR reaction, and the size of the insert was determined by gel electrophoresis. In addition, DNA sequencing was performed on some PCR products.
  • the PCR product obtained in Example 2 was spotted on a glass substrate after being separated and processed, and a genomic chip was obtained after drying.

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Abstract

L'invention concerne un procédé d'amplification par PCR, selon lequel un chromosome artificiel ou un grand fragment d'ADN d'une longueur de 50-5000 kb est utilisé en tant que matrice, l'amorce utilisée étant spécifique d'une séquence Alu. Ladite amorce spécifique d'une séquence Alu se fixe spécifiquement à l'extrémité 5' d'une séquence Alu et s'étend de l'extrémité 3' à l'extrémité 5' de la séquence Alu, ou bien, ladite amorce se fixe spécifiquement à l'extrémité 3' d'une séquence Alu et s'étend de l'extrémité 5' à l'extrémité 3' de la séquence Alu. L'invention concerne également la mise en oeuvre dudit procédé pour la préparation de puces génomiques. Dans les produits d'amplification obtenus selon ce procédé, la séquence de répétition Alu peut être éliminée, ainsi un signal de fond non spécifique d'une puce génomique préparée selon ledit procédé peut être réduit de façon extrême.
PCT/CN2001/001208 2001-07-27 2001-07-27 Nouveau procede d'elimination d'une sequence de repetition dans le genome, et sa mise en oeuvre pour la preparation de puces genomiques Ceased WO2003014384A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CNB018236065A CN1288251C (zh) 2001-07-27 2001-07-27 一种消除基因组重复序列的新方法及其在基因组芯片中的应用
PCT/CN2001/001208 WO2003014384A1 (fr) 2001-07-27 2001-07-27 Nouveau procede d'elimination d'une sequence de repetition dans le genome, et sa mise en oeuvre pour la preparation de puces genomiques

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PCT/CN2001/001208 WO2003014384A1 (fr) 2001-07-27 2001-07-27 Nouveau procede d'elimination d'une sequence de repetition dans le genome, et sa mise en oeuvre pour la preparation de puces genomiques

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773649A (en) * 1996-06-10 1998-06-30 Centre De Recherche De L'hopital Sainte-Justine DNA markers to detect cancer cells expressing a mutator phenotype and method of diagnosis of cancer cells
WO2000022164A1 (fr) * 1998-10-15 2000-04-20 Genset Sondes fluorescentes de peinture chromosomique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773649A (en) * 1996-06-10 1998-06-30 Centre De Recherche De L'hopital Sainte-Justine DNA markers to detect cancer cells expressing a mutator phenotype and method of diagnosis of cancer cells
WO2000022164A1 (fr) * 1998-10-15 2000-04-20 Genset Sondes fluorescentes de peinture chromosomique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Proc. Natl. Acad. Sci, USA, 1996; Vol. 93, No. 9: pages 4474 to 4479 *

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CN1288251C (zh) 2006-12-06
CN1545560A (zh) 2004-11-10

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