CN108410965A - A kind of genetic test probe and gene tester - Google Patents
A kind of genetic test probe and gene tester Download PDFInfo
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- CN108410965A CN108410965A CN201810171949.6A CN201810171949A CN108410965A CN 108410965 A CN108410965 A CN 108410965A CN 201810171949 A CN201810171949 A CN 201810171949A CN 108410965 A CN108410965 A CN 108410965A
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- 238000012360 testing method Methods 0.000 title claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000002372 labelling Methods 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 239000002299 complementary DNA Substances 0.000 claims abstract description 12
- 238000000246 agarose gel electrophoresis Methods 0.000 claims abstract description 7
- 238000001574 biopsy Methods 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
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- 238000012163 sequencing technique Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 58
- 229960002685 biotin Drugs 0.000 claims description 29
- 235000020958 biotin Nutrition 0.000 claims description 29
- 239000011616 biotin Substances 0.000 claims description 29
- 108020004414 DNA Proteins 0.000 claims description 28
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 26
- 210000004027 cell Anatomy 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 22
- 108020004999 messenger RNA Proteins 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 17
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- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
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- 108010017826 DNA Polymerase I Proteins 0.000 claims description 6
- 108020003215 DNA Probes Proteins 0.000 claims description 6
- 239000003298 DNA probe Substances 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 101710128747 Hemoglobin subunit alpha-A Proteins 0.000 claims description 6
- 102100021519 Hemoglobin subunit beta Human genes 0.000 claims description 6
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- 102100034343 Integrase Human genes 0.000 claims description 6
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- 125000004057 biotinyl group Chemical group [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 6
- 230000007850 degeneration Effects 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 6
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- 238000007254 oxidation reaction Methods 0.000 claims description 6
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 6
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
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- 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
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
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Abstract
The present invention relates to technical field of gene detection, especially a kind of genetic test probe includes the cDNA probes containing detection target gene, with specific markers.The invention also discloses a kind of gene testers using genetic test probe, include the following steps：S1, target cell, 1 1.5g of tissue biopsy are collected, is resuspended with 7 9mL deionized waters with 7 9mL, multigelation smudge cells, supernatant is collected after centrifugation, extract sample rna；S2, structure PCR reaction systems, include the sample rna 1ul of extraction, 2 3.5ul of upstream and downstream primer and probe mixed liquor, PCR enzyme mixations 3 6ul of 0.3 0.8ul, MasterMix, carry out PCR amplification；S3, into row agarose gel electrophoresis and sequencing is delivered.The present invention proposes a kind of genetic test probe, can aid in genetic test, and proposes corresponding labeling method, is simply easily achieved, and is suitble to promote.
Description
Technical field
The present invention relates to technical field of gene detection more particularly to a kind of genetic test probes and gene tester.
Background technology
Gene probe, i.e. nucleic acid probe are one section with detection label, and known to sequence, with target gene complementation
Nucleic acid sequence (DNA or RNA).Gene probe is hybridized by molecule to be combined with target gene, generates hybridization signal, can be from great writing brush
Target gene is shown in genome.According to Hybridization principle, the nucleic acid sequence as probe must at least have following two
A condition：1. should be single-stranded, if double-strand, it is necessary to leading denaturation treatment.2. should carry and be easy detected label.It can be wrapped
Whole gene is included, can also be only a part for gene；Can be DNA itself, can also be by transcription from RNA.
Genetic test is the technology being detected to DNA by blood, other body fluid or cell, is taken outside detected person
All venous blood or other histocytes, after expanding its gene information, by particular device to the DNA molecular in detected person's cell
Information detects, and analyzes gene type contained by it and gene defect and its a kind of whether normal method of expressive function, from
And it allows one to understand the gene information of oneself, specify the cause of disease or predicts the risk that body suffers from certain disease.
Medical diagnosis on disease is to cause the mutator of genetic disease with technique of gene detection detection.It is most widely used at present
Genetic test is the auxiliary diagnosis of the detection of newborn's genetic disease, the diagnosis of genetic disease and certain common diseases.Gene is visited
Needle can be used in genetic test, but lack the method that gene probe is marked in the prior art.
Invention content
The purpose of the present invention is to solve disadvantage existing in the prior art, and a kind of genetic test probe that proposes and
Gene tester.
To achieve the goals above, present invention employs following technical solutions：
A kind of genetic test probe is designed, includes the cDNA probes containing detection target gene, with specific markers.
Preferably, preparation method is as follows：Corresponding mRNA, Zhi Hou are isolated and purified from target cell, tissue first
Under the action of reverse transcriptase, DNA, that is, cDNA complementary therewith is synthesized, adds specific markers later.
Preferably, target cell, be organized as hematopoietic cell, corresponding mRNA is α or beta globin mRNA.
Preferably, the method for adding specific markers is as follows, is made in DNA probe double-strand with the DNAse of debita spissitudo I first
At notch, then the nucleosides with 5 ' phosphoric acid is cut by means of 5 ' → 3 ' 5 prime excision enzyme activity of DNa poly meras I again
Acid；The complementary nucleotide that 32P is marked is set to fill into notch using 5 ' → 3 ' poly- enzymatic activitys of the enzyme again simultaneously, archaeal dna polymerase I
Both active alternating actions make notch constantly be moved to 3 ' direction, while the nucleotide in DNA chain is constantly that 32P is marked
The nucleotide of note is replaced.
Preferably, the method for adding specific markers is, using T4 RNA ligases, biotin labeling is held the 3 ' of RNA,
Laeger etc. puts on biotin by nick-translation under enzyme effect, using the UTP analogs containing biotinyl, in enzyme
Promote under reaction, by biotin incorperation DNA.
Preferably, the method for adding specific markers is, with the chemistry route of periodate oxidation, biotin to be connected to
3 ' the ends of RNA, or make with glutaraldehyde the albumen H of the group of biotin labeling, and in single-stranded core crosslinking.
The invention also discloses a kind of gene testers using genetic test probe, include the following steps：
S1, target cell, tissue biopsy 1-1.5g are collected, is resuspended with 7-9mL 7-9mL deionized waters, multigelation is broken thin
Supernatant is collected after centrifugation in born of the same parents, extracts sample rna；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 2-3.5ul,
PCR enzyme mixations 0.3-0.8ul, MasterMix 3-6ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
Preferably, the reaction condition of PCR amplification is as follows：85-92 DEG C of pre-degeneration 10min, subsequently into following cycle：85-
92 DEG C of 45s, 48-52 DEG C of denaturation 45s, 70-75 DEG C of annealing extension 55s, totally 32 cycles, extend in 72 DEG C after circulation terminates
8min, 3 DEG C of preservations.
Preferably, deposition condition is in S3：2% Ago-Gel, 100V voltages, time 35min.
A kind of genetic test probe proposed by the present invention and gene tester, advantageous effect are：The present invention proposes
A kind of genetic test probe, can aid in genetic test, and propose corresponding labeling method, simply be easily achieved, and be suitble to
It promotes.
Specific implementation mode
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment 1
A kind of genetic test probe includes the cDNA probes containing detection target gene, with specific markers.
Preparation method is as follows：Corresponding mRNA is isolated and purified from target cell, tissue first, later in reverse transcriptase
Under the action of, DNA, that is, cDNA complementary therewith is synthesized, adds specific markers later.
Target cell is organized as hematopoietic cell, and corresponding mRNA is α or beta globin mRNA.
The method for adding specific markers is as follows, causes notch in DNA probe double-strand with the DNAse of debita spissitudo I first,
Then the nucleotide with 5 ' phosphoric acid is cut by means of 5 ' → 3 ' 5 prime excision enzyme activity of DNa poly meras I again；Simultaneously again
Using 5 ' → 3 ' poly- enzymatic activitys of the enzyme, the complementary nucleotide that 32P is marked is made to fill into notch, both activity of archaeal dna polymerase I
Alternating action, make notch constantly to 3 ' direction move, while the nucleotide in DNA chain constantly be 32P label nucleotide
Replaced.
The method of addition specific markers is, using T4 RNA ligases, by biotin labeling in the 3 ' ends of RNA, Laeger
Deng by nick-translation, biotin is put under enzyme effect, using the UTP analogs containing biotinyl, in enzymatic reaction
Under, by biotin incorperation DNA.
The method for adding specific markers is, with the chemistry route of periodate oxidation, biotin to be connected to the 3 ' of RNA
End, or make with glutaraldehyde the albumen H of the group of biotin labeling, and in single-stranded core crosslinking.
The invention also discloses a kind of gene testers using genetic test probe, include the following steps：
S1, target cell, tissue biopsy 1g are collected, is resuspended with 7mL 7mL deionized waters, multigelation smudge cells, after centrifugation
Supernatant is collected, sample rna is extracted；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 2ul, PCR enzyme
Mixed liquor 0.3ul, MasterMix 3ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
The reaction condition of PCR amplification is as follows：85 DEG C of pre-degeneration 10min, subsequently into following cycle：85 DEG C of denaturation 45s, 48
DEG C annealing 45s, 70 DEG C extension 55s, totally 32 cycle, after circulation terminates in 72 DEG C extend 8min, 3 DEG C preservation.
Deposition condition is in S3：2% Ago-Gel, 100V voltages, time 35min.
Embodiment 2
A kind of genetic test probe includes the cDNA probes containing detection target gene, with specific markers.
Preparation method is as follows：Corresponding mRNA is isolated and purified from target cell, tissue first, later in reverse transcriptase
Under the action of, DNA, that is, cDNA complementary therewith is synthesized, adds specific markers later.
Target cell is organized as hematopoietic cell, and corresponding mRNA is α or beta globin mRNA.
The method for adding specific markers is as follows, causes notch in DNA probe double-strand with the DNAse of debita spissitudo I first,
Then the nucleotide with 5 ' phosphoric acid is cut by means of 5 ' → 3 ' 5 prime excision enzyme activity of DNa poly meras I again；Simultaneously again
Using 5 ' → 3 ' poly- enzymatic activitys of the enzyme, the complementary nucleotide that 32P is marked is made to fill into notch, both activity of archaeal dna polymerase I
Alternating action, make notch constantly to 3 ' direction move, while the nucleotide in DNA chain constantly be 32P label nucleotide
Replaced.
The method of addition specific markers is, using T4 RNA ligases, by biotin labeling in the 3 ' ends of RNA, Laeger
Deng by nick-translation, biotin is put under enzyme effect, using the UTP analogs containing biotinyl, in enzymatic reaction
Under, by biotin incorperation DNA.
The method for adding specific markers is, with the chemistry route of periodate oxidation, biotin to be connected to the 3 ' of RNA
End, or make with glutaraldehyde the albumen H of the group of biotin labeling, and in single-stranded core crosslinking.
The invention also discloses a kind of gene testers using genetic test probe, include the following steps：
S1, target cell, tissue biopsy 1.2g are collected, is resuspended with 8mL 8mL deionized waters, multigelation smudge cells, centrifugation
After collect supernatant, extract sample rna；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 2.5ul, PCR
Enzyme mixation 0.4ul, MasterMix 4ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
The reaction condition of PCR amplification is as follows：88 DEG C of pre-degeneration 10min, subsequently into following cycle：86 DEG C of denaturation 45s, 49
DEG C annealing 45s, 72 DEG C extension 55s, totally 32 cycle, after circulation terminates in 72 DEG C extend 8min, 3 DEG C preservation.
Deposition condition is in S3：2% Ago-Gel, 100V voltages, time 35min.
Embodiment 3
A kind of genetic test probe includes the cDNA probes containing detection target gene, with specific markers.
Preparation method is as follows：Corresponding mRNA is isolated and purified from target cell, tissue first, later in reverse transcriptase
Under the action of, DNA, that is, cDNA complementary therewith is synthesized, adds specific markers later.
Target cell is organized as hematopoietic cell, and corresponding mRNA is α or beta globin mRNA.
The method for adding specific markers is as follows, causes notch in DNA probe double-strand with the DNAse of debita spissitudo I first,
Then the nucleotide with 5 ' phosphoric acid is cut by means of 5 ' → 3 ' 5 prime excision enzyme activity of DNa poly meras I again；Simultaneously again
Using 5 ' → 3 ' poly- enzymatic activitys of the enzyme, the complementary nucleotide that 32P is marked is made to fill into notch, both activity of archaeal dna polymerase I
Alternating action, make notch constantly to 3 ' direction move, while the nucleotide in DNA chain constantly be 32P label nucleotide
Replaced.
The method of addition specific markers is, using T4 RNA ligases, by biotin labeling in the 3 ' ends of RNA, Laeger
Deng by nick-translation, biotin is put under enzyme effect, using the UTP analogs containing biotinyl, in enzymatic reaction
Under, by biotin incorperation DNA.
The method for adding specific markers is, with the chemistry route of periodate oxidation, biotin to be connected to the 3 ' of RNA
End, or make with glutaraldehyde the albumen H of the group of biotin labeling, and in single-stranded core crosslinking.
The invention also discloses a kind of gene testers using genetic test probe, include the following steps：
S1, target cell, tissue biopsy 1.4g are collected, is resuspended with 8mL 8mL deionized waters, multigelation smudge cells, centrifugation
After collect supernatant, extract sample rna；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 3ul, PCR enzyme
Mixed liquor 0.7ul, MasterMix 5ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
The reaction condition of PCR amplification is as follows：90 DEG C of pre-degeneration 10min, subsequently into following cycle：90 DEG C of denaturation 45s, 50
DEG C annealing 45s, 74 DEG C extension 55s, totally 32 cycle, after circulation terminates in 72 DEG C extend 8min, 3 DEG C preservation.
Embodiment 4
A kind of genetic test probe includes the cDNA probes containing detection target gene, with specific markers.
Preparation method is as follows：Corresponding mRNA is isolated and purified from target cell, tissue first, later in reverse transcriptase
Under the action of, DNA, that is, cDNA complementary therewith is synthesized, adds specific markers later.
Target cell is organized as hematopoietic cell, and corresponding mRNA is α or beta globin mRNA.
The method for adding specific markers is as follows, causes notch in DNA probe double-strand with the DNAse of debita spissitudo I first,
Then the nucleotide with 5 ' phosphoric acid is cut by means of 5 ' → 3 ' 5 prime excision enzyme activity of DNa poly meras I again；Simultaneously again
Using 5 ' → 3 ' poly- enzymatic activitys of the enzyme, the complementary nucleotide that 32P is marked is made to fill into notch, both activity of archaeal dna polymerase I
Alternating action, make notch constantly to 3 ' direction move, while the nucleotide in DNA chain constantly be 32P label nucleotide
Replaced.
The method of addition specific markers is, using T4 RNA ligases, by biotin labeling in the 3 ' ends of RNA, Laeger
Deng by nick-translation, biotin is put under enzyme effect, using the UTP analogs containing biotinyl, in enzymatic reaction
Under, by biotin incorperation DNA.
The method for adding specific markers is, with the chemistry route of periodate oxidation, biotin to be connected to the 3 ' of RNA
End, or make with glutaraldehyde the albumen H of the group of biotin labeling, and in single-stranded core crosslinking.
The invention also discloses a kind of gene testers using genetic test probe, include the following steps：
S1, target cell, tissue biopsy 1g are collected, is resuspended with 9mL 9mL deionized waters, multigelation smudge cells, after centrifugation
Supernatant is collected, sample rna is extracted；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 3.5ul, PCR
Enzyme mixation 0.8ul, MasterMix 6ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
The reaction condition of PCR amplification is as follows：85 DEG C of pre-degeneration 10min, subsequently into following cycle：85 DEG C of denaturation 45s, 48
DEG C annealing 45s, 70 DEG C extension 55s, totally 32 cycle, after circulation terminates in 72 DEG C extend 8min, 3 DEG C preservation.
Deposition condition is in S3：2% Ago-Gel, 100V voltages, time 35min.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of genetic test probe, which is characterized in that include the cDNA probes containing detection target gene, with specific mark
Note.
2. a kind of genetic test probe according to claim 1, which is characterized in that preparation method is as follows：First from mesh
Corresponding mRNA is isolated and purified in mark cell, tissue, later under the action of reverse transcriptase, synthesizing DNA complementary therewith is
CDNA adds specific markers later.
3. a kind of genetic test probe according to claim 2, it is characterised in that：Target cell is organized as hematopoietic cell,
Corresponding mRNA is α or beta globin mRNA.
4. a kind of genetic test probe according to claim 2, it is characterised in that：The method for adding specific markers is as follows,
Notch is caused in DNA probe double-strand with the DNAse of debita spissitudo I first, then again by means of the 5 ' of DNa poly meras I
→ 3 ' 5 prime excision enzyme activity cuts the nucleotide with 5 ' phosphoric acid；Make 32P using 5 ' → 3 ' poly- enzymatic activitys of the enzyme again simultaneously
The complementary nucleotide of label fills into notch, both active alternating actions of archaeal dna polymerase I, make notch constantly to 3 ' side
The nucleotide that nucleotide to movement, while in DNA chain is constantly marked by 32P replaces.
5. a kind of genetic test probe according to claim 2, it is characterised in that：The method for adding specific markers is to make
With T4RNA ligases, by biotin labeling at the 3 ' ends of RNA, Laeger etc. puts on life by nick-translation under enzyme effect
Object element, using the UTP analogs containing biotinyl, under enzymatic reaction, by biotin incorperation DNA.
6. a kind of genetic test probe according to claim 2, it is characterised in that：The method of addition specific markers is to use
Biotin, is connected to the 3 ' ends of RNA by the chemistry route of periodate oxidation, or makes with glutaraldehyde the egg of the group of biotin labeling
On white H, with single-stranded core crosslinking.
7. a kind of gene tester using genetic test probe according to claim 1, which is characterized in that including with
Lower step：
S1, target cell, tissue biopsy 1-1.5g are collected, is resuspended with 7-9mL 7-9mL deionized waters, multigelation is broken thin
Supernatant is collected after centrifugation in born of the same parents, extracts sample rna；
S2, structure PCR reaction systems, include the sample rna 1ul of extraction, upstream and downstream primer and probe mixed liquor 2-3.5ul,
PCR enzyme mixations 0.3-0.8ul, MasterMix 3-6ul carries out PCR amplification；
S3, into row agarose gel electrophoresis and sequencing is delivered.
8. a kind of gene tester using genetic test probe according to claim 6, which is characterized in that PCR expands
The reaction condition of increasing is as follows, 85-92 DEG C of pre-degeneration 10min, subsequently into following cycle：85-92 DEG C of 45s, 48-52 DEG C of denaturation is moved back
Fiery 45s, 70-75 DEG C of extension 55s, totally 32 cycles, extend 8min, 3 DEG C of preservations in 72 DEG C after circulation terminates.
9. a kind of gene tester using genetic test probe according to claim 6, which is characterized in that electric in S3
Swimming condition is：2% Ago-Gel, 100V voltages, time 35min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1270229A (en) * | 2000-03-28 | 2000-10-18 | 陈高明 | Polygenic amplification chip and preparing process thereof |
| CN1635143A (en) * | 2003-12-31 | 2005-07-06 | 中国科学院上海生命科学研究院 | Down-regulated genes expressed in liver cancer cells and their application |
-
2018
- 2018-03-01 CN CN201810171949.6A patent/CN108410965A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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