CN1545559A - A novel method for eliminating repeat sequence in genome and its use in preparing of FISH probe - Google Patents
A novel method for eliminating repeat sequence in genome and its use in preparing of FISH probe Download PDFInfo
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- CN1545559A CN1545559A CNA018236057A CN01823605A CN1545559A CN 1545559 A CN1545559 A CN 1545559A CN A018236057 A CNA018236057 A CN A018236057A CN 01823605 A CN01823605 A CN 01823605A CN 1545559 A CN1545559 A CN 1545559A
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- alu
- sequence
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- specific primers
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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
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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
- C12Q1/6841—In situ hybridisation
Abstract
The present invention discloses a method for amplification by polymerase chain reaction, in which an artificial chromosome or a large DNA fragment of 50-5000kb in length is used as a template, and an Alu-specific primer is used as a primer. Said Alu-specific primer binds specifically to 5'-terminus of an Alu sequence, and extends from 3' to 5' of the Alu sequence, alternatively, said primer binds specifically to 3'-terminus of an Alu sequence, and extends from 5' to 3' of the Alu sequence. The present invention also includes the use of said method in preparing of genome chip. The amplification products obtained by the present method can eliminate the Alu repeat sequence, so non-specific background signal of a FISH probe prepared by said method can be decreased dramatically.
Description
A kind of new method for eliminating genome repetitive sequence and its applied technical field in FISH probe preparation
The present invention relates to the new method that genome repetitive sequence is eliminated during FISH probes are prepared using artificial chromosome.Genome repetitive sequence is not contained in the genomic DNA sequence obtained using the new method, therefore effectively eliminates the nonspecific hybridization signals caused by repetitive sequence, the accuracy of FISH probe is improved.Background technology
In June, 2000, the Human Genome Project discloses " working draft " of human genome.On January 15th, 2000, " formal version " human genome map has also emerged.As human genome is gradually decrypted, the figure of a life will be drawn, and great variety will also occur for the life of people.Developing rapidly for the Human Genome Project provides new chance for the exploitation of biotechnology.
All kinds of artificial chromosomes containing large fragment DNA molecule are to develop one of byproduct of the Human Genome Project.Artificial chromosome includes yeast artificial chromosome（Yeast Artficial Chromosome, YAC.Its Insert Fragment is about 1,000 kilobase pair）, bacteriophage artificial chromosome（Phage Artificial Chromosome, PAC.Its Insert Fragment is about 100 kilobase pair）, bacterial artificial chromosome (Bacterial Artificial Chromosome, BAC.Its Insert Fragment is about 100 kilobase pair）.
This kind of artificial chromosome can be used as chromosome fluorescence in-situ hybridization（Fluorescence In Site Hybridization, FISH) probe, for multiple use, including genetic diseases diagnosis（Mainly various chromosome abnormalities）, pre-natal diagnosis（Pregnant woman's hereditary disease sieves Check）, tumor classification, diagnosis and prognosis inspection, observation radioactive ray and infringement of other environmental factors to human body.Further, since most of these artificial chromosomes oneself be positioned, so they can be used to make genome chip.
There is the DNA sequence dna of many repetitions in human genome.These sequences are closely similar, continuously emerged in whole gene group.Wherein, with Alu sequence frequency of occurrences highest, per every about 4 kilobase to just occurring once.Alu sequence accounts for human genome STb gene amount 5% or so, up to 900,000 copy, wherein 60% sequence member contains restriction enzyme A lu otch, therefore it is called Alu family, the R bands in cell division late period are concentrated mainly on, largely belong to noncoding DNA, but also some be located at rnRNA non-translational region, even in code area.Alu sequence most long about 150 base-pairs.Mankind Alu
Sequence length about 300bp, itself is made up of 120bp and 150bp repetitive sequence, separated between the two by the region rich in A, there is one section of 7- 10bp direct repetitive sequence at two ends again again.Between Alu sequence not fully, there are some differences.Due to these repetitive sequences can phase mutual cross and produce non-specific background signal, thus Specific hybridization signals can be disturbed.This is that manually chromosome makes the biggest problem that FISH probe is faced at present.
Therefore, this area eliminates the technology of Alu repeated sequence in the urgent need to Jian is sent out, to obtain the FISH probe of reduction unspecific background signal.The content of the invention
It is an object of the invention to provide a kind of method of new amplification human gene group DNA, this method is used to effectively remove repetitive sequence when expanding human gene group DNA's sequence in artificial chromosome, so as to eliminate the interference of the nonspecific hybridization signals caused by repetitive sequence, the accuracy and reliability of results of hybridization are improved.
In the first aspect of the present invention, there is provided a kind of PCR amplification method, wherein, it is template by 50-5000kb large fragment DNA molecule of artificial chromosome or length, and using Alu specific primers as primer, described Alu specific primers are all specifically incorporated into the 5' ends of Alu sequence and bearing of trend and are 3' -5' directions of Alu sequence, or be all incorporated into the 3' ends of Alu sequence and bearing of trend be Alu sequence 5' -3' directions.
In the preference of the present invention, described Alu specific primers length is 15-25bp.In another-preferred, described Alu specific primers are selected from the group：
Alu-Nl : 5--TTA CAG GYR TCA GCC ACY AC-3* (SEQ ID N0 : 1)
Alu-N2 : 5'-RCC AYT GCA CTY CAG CCT G- 3' (SEQ ID NO : 2)
Wherein：Y=C or T;1^ or 6.
In the second aspect of the present invention there is provided a kind of method for producing fluorescence labeling probe, it includes step：
(a) polynucleotide products are gone out by PCR amplification, by 50-5000kb large fragment DNA molecule of artificial chromosome or length it is template in the polymerase chain reaction, and using Alu specific primers as primer, described Alu specific primers are all specifically incorporated into the 5' ends of Alu sequence and bearing of trend is 3' → 5' directions of Alu sequence, or are all incorporated into 3' ends and the bearing of trend of Alu sequence
For 5' → 3' directions of Alu sequence；
(b) to step（A) polynucleotide products in carry out fluorescence labeling, produce fluorescence labeling probe.In the preference of the present invention, described fluorescence labeling probe is fluorescence in situ hybridization probe.In the third aspect of the present invention, obtained fluorescence labeling probe in aforementioned manners is additionally provided.
Brief description of the drawings
Fig. 1 is the electrophoretogram of the PCR primer expanded with Alu primers, and wherein template is the DNA of BAC clones.In figure, molecular weight marker（M) be lkb DNA ladder（DNA ladder).The grades of RPII-497 I 24 are the numbering that BAC is cloned, and wherein RPII is BAC clone libraries（It is made up of many plates）Title, the set of number after " RP11- "（Title as 497) being one of plate in the library, followed by English alphabet（Such as I) and another set of number（As 24) referred to abscissa and ordinate that the BAC is cloned in present position in this disk respectively.
5' in figure represents 5' primers, i.e. Alu-Nl: 5'- TTA CAG GYR TCA GCC ACY AC- 3 (SEQ ID N0 : 1)；3' represents 3' primers, g Jie Alu_N2： 5'- RCC AYT GCA CTY CAG CCT G- 3' (SEQ ID NO :2), wherein：The C of Y bis- or T; 1=eight or 6.
Fig. 2 is the Southern trace figures of the PCR primer expanded with Alu primers.Wherein molecular weight marker (M) is lkb DNA ladder (DNA ladder).Positive control 1 and 2 (CTRL1 and CTRL2) is BAC DNA.In figure, what it is as probe is that whole BAC clones DNA.The numbering of BAC clones is RPII- 110-0-7, and size is 120kb.
Embodiment
As used herein, term " artificial chromosome ", which refers to, artificial constructed has chromosomal characteristics（Such as contain centromere, telomere and DNA replication dna starting point, while also being marked with transformation and selection）, can self-replacation carrier.Representational artificial chromosome includes but is not limited to：Yeast artificial chromosome（Yeast Artficial Chromosome, YAC), bacteriophage artificial chromosome（Phage Artificial Chromosome, PAC), bacterial artificial chromosome (Bacterial Artificial Chromosome, BAC), artificial mammalian chromosome（Mammalian Artificial Chromosome, MAC).For example, YAC uses yeast chromosomal centromere, telomere, the rest may be inferred.
As used herein, term " Alu specific primers " refers to the end for specifically binding to Alu sequence, so that amplified product of polymerase chain reaction does not contain or contain substantially no the primer of Alu repeated sequence.Alu specific primers are divided into two classes：First kind Alu specific primers be specifically be incorporated into Alu sequence 5' ends and bearing of trend be Alu sequence 3' -5' directions；Equations of The Second Kind Alu specific primers be incorporated into Alu sequence 3' ends and bearing of trend be Alu sequence 5' → 3' directions.The present inventor establishes a kind of new method for the efficient amplification human DNA sequence in artificial chromosome by in-depth study extensively.The process employs design unique amplimer and amplification condition, wherein amplimer is at the 5' ends and 3' ends of the Alu repeated sequence occurred positioned at human gene group DNA's high frequency.The characteristics of this method is to expand human gene group DNA's sequence in such as bacterial artificial chromosome (BAC) by PCR method using this primer, can effectively remove the Alu repeated sequence of human gene group DNA's high frequency appearance.
The principle of this technology method is the characteristics of utilizing the Alu sequence frequency of occurrences, using the sequence at Alu-end as template, to design corresponding primer.When two Alu sequences be separated by it is not far, i.e., in the range of a normal PCR reaction amplification（It is general internal in 2 kilobase）And the direction of the two Alu sequences is relative（If 5' ends are to 5' ends）When, it is possible to amplify the genomic dna sequence between this two Alu sequences.Because the DNA sequence dna amplified with this method is located between two Alu repeated sequences, so it no longer contains repetitive sequence.
By taking bacterial artificial chromosome (BAC) as an example, its size is typically in 100 kilobase to left and right.Calculated with averagely every 4 kilobase there is an Alu sequence, can generally have the Alu sequence of 25 or so in a BAC, wherein quite a few standoff distance is in the range of PCR reaction amplifications.Therefore, as the human gene group DNA in using the Alu special primers of present invention amplification BAC, it is possible to greatly increase the chance for obtaining unique sequence.
In the present invention, PCR reaction conditions are not particularly limited, the PCR conditions of conventional specific amplification can be used in the present invention.A kind of conventional condition is that 90-95 is denatured 45-75 seconds, and 50-65 V anneal 30-90 seconds, and 70-74 °C extends 30-90 seconds, common 25-35 circulation.
The results show feasibility and high efficiency of this novel technical method.The present inventor have chosen 100 BAC clones for being from No. 3 chromosomes of people and No. 16 chromosomes, and the Alu sequence 5' ends designed respectively with the present invention and 3' terminal specifics primer are expanded.All BAC obtain at least one PCR primer, at most
Can obtain ten several PCR primers.
In addition, also making probe from a BAC containing a variety of repetitive sequences（RP-11- 110-0-7), through radio isotope32After P marks molecule hybridization reaction is carried out with these PCR primers.Reaction result shows：All PCR primers are free of repetitive sequence.
Determined dna sequence analysis further has been done to some PCR primers.Sequencing result also demonstrates that the size of these PCR products between 400-2000 base-pairs, average about 800 base-pairs, and is all free of repetitive sequence.
Therefore, single human gene group DNA's sequence without repetitive sequence quickly can be amplified from the artificial chromosomes such as BAC clones or large fragment DNA using the novel technical method of the present invention.The present invention technology have multiple use, including but be not limited to：
(1) these be amplified obtained unique sequence and can carry out Southern microsphere hybridization as gene probe；
(2) these be amplified obtained unique sequence and can pass through fluorescence labeling, carry out chromosome fluorescence in-situ hybridization（FISH) ;
(3) these be amplified obtained unique sequence and can combine genome DNA microarray is made in surface of solid phase carriers, carry out genetic test and diagnosis.The oligonucleotides or its fragment obtained with the inventive method can be used for preparing probe, such as chromosome fluorescence in-situ hybridization（FISH) probe.Therefore, present invention also offers a kind of method for producing fluorescence labeling probe, especially FISH probe.The method difference with the prior art that the present invention prepares FISH probe is only that the DNA as probe is different.Therefore, the method and fluorescent dye of conventional various preparation FISH probes（Such as fluorescein, rhodamine）It can be used in the present invention.
Alu repeated sequence is eliminated due to obtaining amplified production with the inventive method, therefore, can be significantly reduced with the unspecific background signal of its FISH probe prepared.With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition such as Sambrook et al., molecular cloning：Laboratory manual（New York:
Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Embodiment 1
Primer is synthesized
The method manually synthesized is respectively synthesized following primer：
Alu-Nl : 5'- TTA CAG GYR TCA GCC ACY AC- 3' (SEQ ID N0 : 1)
Alu-N2 : 5'-RCC AYT GCA CTY CAG CCT G- 3' (SEQ ID NO : 2)
Wherein：Y=C or T;R sixteen or..Embodiment 2
With Alu primer amplifieds bacterial artificial chromosome (BAC)
1. cultivates bacterial artificial chromosome (BAC) clone, is coated with culture medium flat plate, and single clone is selected in culture, and the Shaking culture clone bacterium extracts BAC DNA from the bacterium.
2. PCR react BAC DNA fragmentation of the amplification without repetitive sequence method it is following-take the 50 ng BAC DNA (templates reacted as PCR）, 50 μ 1 (microlitre）Reacted in reaction cumulative volume.Composition in the reaction system is：The raM primer Alu- of the mM of 10 raM Tris-HCl, pH 8. 4,2 raM MgCl, 50 mM KCl, 0. 1 gelatin, 200 mM Dntp, 0. 5 Nl or Alu-N2,2 unit Taq polymerase；).PCR reaction condition be：94 °C 1 minute, 60 °C 1 minute, 72 °C 2 minutes, 30 circulation.
It is as shown in Figure 1 for the electrophoresis result that template enters performing PCR reaction with different BAC clones.PCR primer average out to 2- 5.Embodiment 3
To the Southern engram analysis of PCR primer
By the PCR primer obtained in embodiment 2 after gel electrophoresis, it is transferred on nylon membrane.With32Complete BAC clones labeled P（RP11- 110- 0- 7) DNA is as probe, with Southern blotting membranes hybridization.
Southern blot analysis results are as shown in Figure 2.Because probe used（Complete BAC) in contain
There is substantial amounts of repetitive dna sequence, and the PCR primer amplified with Alu primers does not hybridize, therefore be free of repetitive dna sequence in this explanation PCR primer.Embodiment 4
Preservation, screening and identification to PCR primer
The pcr amplification product of above-mentioned acquisition is cloned into TA- Vector carriers, in order to preserve and massive duplication from now on.
Then, negative clone is screened with Alu sequence.RPII-110- 0-7 (size is 120kb) are cloned with bacterial artificial chromosome filter out the negative clone without Alu repeated sequence.
Then, Insert Fragment is amplified with PCR reactions, and passes through the size of gel electrophoresis therapy determining Insert Fragment.In addition, also further having done determined dna sequence analysis to some PCR primers.
Sequencing result also demonstrates that the size of these PCR primers between 400-2000 base-pairs, average about 800 base-pairs, and is all free of repetitive sequence.Embodiment 5
The preparation of FISH probe
In the present embodiment, with the PCR primer prepared in above-described embodiment with Alu specific primers, FISH probe is prepared by conventional random primering.The method carrys out marker DNA molecule using random primer, and the label probe of initial amount of DNA is several times as much as by marking reaction to produce.The random primer labelling kit of Life Technologies companies can typically be used.
(1) lOOmg DNA are dissolved in the water of 24 μ 1, is placed on ice, adds the random primer solution of 20 μ 1 (2. 5 Χ).By denatured by boiling 5 minutes of mixed DNA, it is immediately placed on ice.
(2) the lOXdNTP solution of 5 μ 1 is added, lightly reagent in centrifuge tube is thoroughly mixed, Ι μ enzyme solutions are added.
(3) mix after hooking, reactant is cultivated 1 hour in 37 °C, take 2 μ 1 to be gel electrophoresis inspection Check.If labeled DNA probe fragment is between 200-2000bp, best results are marked.
(4) terminating reaction can be by being heated to 75,10 minutes, or adds the reaction terminating buffer solutions of 5 μ 1.Obtain FISH probe.
Claims (1)
- Claims1. a kind of PCR amplification method, it is characterized in that, it is template by 50-5000kb large fragment DNA molecule of artificial chromosome or length, and using Alu specific primers as primer, described Alu specific primers are all specifically incorporated into the 5' ends of Alu sequence and bearing of trend and are 3' -5' directions of Alu sequence, or be all incorporated into the 3' ends of Alu sequence and bearing of trend be Alu sequence 5' -3' directions.2. the method as described in claim 1, it is characterised in that the artificial chromosome is selected from the group：Yeast artificial chromosome, bacteriophage artificial chromosome, bacterial artificial chromosome and artificial mammalian chromosome.3. the method as described in claim 1, it is characterised in that described PCR reaction conditions are：90-95 °C is denatured 45-75 seconds, and 50-65 °C is annealed 30-90 seconds, and 70-74 °C extends 30-90 seconds, common 25-35 circulation.4. the method as described in claim 1, it is characterised in that described Alu specific primers length is 15-25bp.5. the method as described in claim 1, it is characterised in that described Alu specific primers are selected from the group：Alu-Nl : 5'-TTA CAG GYR TCA GCC ACY AC-3*(SEQ ID NO: l)Alu-N2: 5'-RCC AYT GCA CTY CAG CCT G-3' (SEQ ID NO:2)Wherein：Y=C or T;1=eight or 0.6.-kind of the method for producing fluorescence labeling probe, it is characterised in that it includes step：(a) polynucleotide products are gone out by PCR amplification, by 50-5000kb large fragment DNA molecule of artificial chromosome or length it is template in the polymerase chain reaction, and using Alu specific primers as primer, described Alu specific primers are all specifically incorporated into the 5' ends of Alu sequence and bearing of trend and are 3' -5' directions of Alu sequence, or be all incorporated into the 3' ends of Alu sequence and bearing of trend be Alu sequence 5' → 3' directions；(b) fluorescence labeling is carried out to the polynucleotide products in step (a), produces fluorescence labeling probe.7. method as claimed in claim 6, it is characterised in that described fluorescence labeling probe is fluorescence in situ hybridization probe.8. method as claimed in claim 6, it is characterised in that the artificial chromosome is selected from the group：Yeast artificial chromosome, bacteriophage artificial chromosome, bacterial artificial chromosome and artificial mammalian chromosome.9. method as claimed in claim 6, it is characterised in that described Alu specific primers length is 15-25bp.10. method as claimed in claim 6, it is characterised in that described Alu specific primers are selected from the group：Alu-Nl : 5'-TTA CAG GYR TCA GCC ACY AC-3'(SEQ ID NO: l)Alu-N2:5 ,-RCC AYT GCA CTY CAG CCT G-3'(SEQ ID NO:2) wherein：Y=C or T;1=eight or 0.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2001/001209 WO2003014385A1 (en) | 2001-07-27 | 2001-07-27 | A novel method for eliminating repeat sequence in genome and its use in preparing of fish probe |
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| CN1545559A true CN1545559A (en) | 2004-11-10 |
| CN1288250C CN1288250C (en) | 2006-12-06 |
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| CNB018236057A Expired - Fee Related CN1288250C (en) | 2001-07-27 | 2001-07-27 | A novel method for eliminating repeat sequence in genome and its use in preparing of FISH probe |
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| WO (1) | WO2003014385A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409504B (en) * | 2013-06-26 | 2015-02-25 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence |
| CN103409506A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting ALK (anaplastic lymphoma kinase) gene free from repetitive sequence |
| CN103409505A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting BCR/ABL fusion gene free from repetitive sequence |
| CN103409507A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, a kit and a detection method for detecting ROS1 gene free from repetitive sequence |
| CN107083417B (en) * | 2016-02-16 | 2020-01-31 | 益善生物技术股份有限公司 | AML1/ETO fusion gene detection kit and detection method |
| CN107083418B (en) * | 2016-02-16 | 2020-08-25 | 益善生物技术股份有限公司 | PML/RAR alpha fusion gene detection kit and detection method |
| CN110205357A (en) * | 2019-06-22 | 2019-09-06 | 福建农林大学 | A kind of preparation method of more primer oligo probes |
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| 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 |
| FR2784683B1 (en) * | 1998-10-15 | 2002-12-13 | Genset Sa | FLUORESCENT PROBES OF CHROMOSOME PAINT |
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- 2001-07-27 WO PCT/CN2001/001209 patent/WO2003014385A1/en active Application Filing
- 2001-07-27 CN CNB018236057A patent/CN1288250C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1288250C (en) | 2006-12-06 |
| WO2003014385A1 (en) | 2003-02-20 |
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