CN104372002A - Internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification - Google Patents
Internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification Download PDFInfo
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Abstract
The invention relates to an internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification, which comprises a pair of specific primers matched with karyon repeated nucleotide segment and a probe, wherein the primer sequences are disclosed as SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is disclosed as SEQ ID NO.3. The internal reference system can be used for quality control on individual cell amplification and calculation on relative number of copies of mitochondrion genes and the like. The elemental form of the system is based on genome DNA PCR and TaqMan fluorescent quantitative PCR; the internal reference is set on the basis of the karyon repeated nucleotide sequence; and the system is suitable for DNA PCR amplification internal references for individual cell and subcellular template quantities, and is especially suitable for rare samples, so that the number of copies of individual cells and other data can be reliable and valuable in deed. The system can be used for fundamental research on individual cells and detection on clinical samples.
Description
Technical field
The present invention relates to a kind of internal reference system that can be used for people's individual cells genome or Mitochondrial DNA pcr amplification, belong to biological technical field.
Background technology
Unicellular gene amplification starts from the nineties in last century.At present, singe-cell PCR amplification is in supplementary reproduction, and the fields such as tumour use widely, and ivf zygote cell function detects, circulating tumor cell Genetic Detection, and these are all the exemplary that unicellular gene amplification detects.Along with high throughput sequencing technologies is research and development and progressively the carrying out of clinical application, it is more and more extensive that unicellular genome amplification detects application.
But in the experiment of routine; we find that individual cells DNA detection usually there will be cell and do not choose; the all or part of loss of cell; the phenomenons such as cracking is incomplete; this just makes internal reference extremely important; otherwise its product amplified, the representativeness of the absolute copy number of gene measured etc. and meaning are just very limited.
Only have two copies, be confined to other internal reference type (as Actin, tubulin etc.) of a chromosomal zonule, although can as internal reference in many cells level, it is then not suitable for doing internal reference at individual cell level, because cannot reflect the incompleteness in other region of cell.When being unicellular reverse transcription RT-PCR, because same RNA can have a lot of copy, so the internal reference of other type also can do the internal reference of single-cell RT-PCR, but is not suitable for the internal reference of unicellular genome or mitochondrial DNA amplification.
Plastosome is the important refinery of cell, energy derive.The plastosome of fetus obtains from mother's ovum substantially.Ovum mitochondrial function directly affects the division of early stage zygote, growth, the growth etc. of embryo.The women of some ovum abnormalities, external supplementary reproduction success ratio is low, moved on to by fertilized egg cell's core in the endochylema of the normal ovum of stoning and then can correct the mitochondrial hereditary defect of ovum, this solves mitochondriopathy, the important method of plastosome serious loss.
Summary of the invention
Applicants have invented a kind of internal reference system for people's individual cells genome or Mitochondrial DNA pcr amplification.
Due to for be individual cells detect; therefore in traditional detection, usually there will be cell do not choose; the all or part of loss of cell; the phenomenons such as cracking is incomplete; this just makes internal reference extremely important; otherwise its product amplified, the representativeness of the absolute copy number of gene measured etc. and meaning are just very limited.General gene only has two copies in individual cells, and the region that is confined to item chromosome very little, be not suitable for and do unicellular or ubcellular DNA cloning internal reference.Because repeated nucleotide sequences has hundreds of thousands of to 1,000,000 copies inside a nucleus, total amount can reach 10% of genome orders, and has extensive distribution at each karyomit(e).Because its copy number is large and more stable, therefore the present invention have selected nucleus repeated nucleotide sequences as the internal reference of unicellular DNA cloning thus for the quality control of people's unicellular DNA pcr amplification, the gene amplification Relative copy number calculating etc. such as plastosome.
Some individual cells source is very limited, and as human oocyte, and a cell usually needs to do much different detections or research, and the repetition of experiment, and this makes the genetic material that can be used for doing single detection well below the genetic material total amount of a cell.Our internal reference system is very suitable for the detection under this subcellsular level.
Internal reference system of the present invention is with unicellular genome amplification PCR or TaqMan quantitative fluorescent PCR for basic form, repeats nucleotide fragments for internal reference with nucleus.Internal reference primer and target primer are with existing with a reaction system, and increase target gene and internal reference fragment respectively simultaneously.In TaqMan pcr amplification, the existence of probe adds the specificity of reaction signal.Unicellular lysate (amplification template) is also comprised, archaeal dna polymerase, amplification substrate, and reaction buffer in reaction system.
In internal reference system of the present invention, comprise a pair and repeat Auele Specific Primer that nucleotide fragments mates and probe with nucleus, its primer sequence is as SEQ ID No.1 and SEQ ID No.2, and probe sequence is as SEQ ID No.3.
As is known to the person skilled in the art, above-mentioned sequence is not absolute, the sequence had with above-mentioned sequence compared with high homologous degree can also be adopted, described two ends conversion or plus-minus 5-10 the nucleotide base referring to above-mentioned sequence compared with high homologous degree, or middle order base has 5-10 individual replaced, or both have.
Internal reference system of the present invention may be used for unicellular amplification kit, this test kit can be used for internal reference for detecting the isogenic Relative copy number of people's individual cells plastosome, or for the Quality Control of unicellular amplification, checking testing sample is with or without collecting individual cells, if any, whether the genetic material of individual cells is complete, and as partial loss, or lysis is incomplete.
Accompanying drawing explanation
Fig. 1 is different people cell amplification template amount internal reference amplification linear graph (0.1-10 Hela cell).
Fig. 2 is that (top is chondriogen amplification, and bottom is reference gene amplification for single human oocyte's plastosome and internal reference qPCR amplification figure.Actual template consumption 0.05 ovum).
Embodiment
Embodiment 1: different people cell amplification template amount internal reference amplification linear relationship
Get growth normal Hela cell 50, cracking (cell pyrolysis liquid by Tris damping fluid, adds SDS and Proteinase K composition) in 10ul cell pyrolysis liquid, after abundant cracking (50 DEG C 50 minutes, 80 DEG C 10 minutes), become every 2ul containing 0.1,0.3,1 with cell lysis buffer, 3,9 cells, as template, do TaqMan qPCR amplification internal reference.After amplification, calculate each reaction group Ct value.Often organize two repetitions.
Experimental result is as shown in following table 1 and accompanying drawing 1.
Table 1: the Ct that different people cell amplification template amount is corresponding
| Template amount (cell count) | Average |
| 9.00 | 17.22 |
| 3.00 | 19.80 |
| 1.00 | 20.43 |
| 0.30 | 21.67 |
| 0.10 | 23.08 |
Embodiment 2: single human oocyte's Mitochondrial DNA and internal reference qPCR increase
Get 10, the single ovum of normal people (from 4 people), abundant cracking in 5ul cell pyrolysis liquid respectively, redilution becomes every 1.5ul containing 0.05 cell, makes template, is TaqMan qPCR and increases, amplification Mitochondrial DNA and internal reference.After amplification, calculate the Ct value of each reaction group plastosome and internal reference respectively.Often organize two repetitions.Experimental result sees the following form shown in 2 and accompanying drawing 2.
Table 2: the Ct that Mitochondrial DNA is corresponding respectively with internal reference
| Plastosome | Internal reference |
| 24.95 | 27.95 |
| 25.54 | 28.03 |
| 26.01 | 28.42 |
| 25.08 | 27.49 |
| 25.58 | 27.09 |
| 26.29 | 27.16 |
| 25.52 | 25.61 |
| 24.87 | 26.80 |
| 26.28 | 26.82 |
| 26.81 | 27.64 |
Claims (4)
1. the internal reference system for people's individual cells genome or Mitochondrial DNA pcr amplification, comprise a pair and repeat Auele Specific Primer that nucleotide fragments mates and probe with nucleus, its primer sequence is as SEQ ID No.1 and SEQ ID No.2, and probe sequence is as SEQ ID No.3.
2. internal reference system according to claim 1, it is characterized in that primer or probe order are the sequence had with above-mentioned sequence compared with high homologous degree, described two ends conversion or plus-minus 5-10 the nucleotide base referring to above-mentioned sequence compared with high homologous degree, or middle order base has 5-10 individual replaced, or both have.
3. internal reference system according to claim 1 and 2, it is characterized in that internal reference is for detecting the isogenic Relative copy number of people's individual cells plastosome, or for the Quality Control of unicellular amplification, checking testing sample is with or without collecting individual cells, if any, whether the genetic material of individual cells is complete, and as partial loss, or lysis is incomplete.
4., according to claim 1,2,3 arbitrary described internal reference systems, is characterized in that the detection kit researched and developed based on this basic skills, and for laboratory study, or clinical sample detects.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410558839.7A CN104372002A (en) | 2014-10-20 | 2014-10-20 | Internal reference system for human individual cell genome or mitochondrion DNA (deoxyribonucleic acid) PCR (polymerase chain reaction) amplification |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116479105A (en) * | 2023-02-22 | 2023-07-25 | 中国疾病预防控制中心职业卫生与中毒控制所 | Nucleic acid reagent, kit and multiplex fluorescence quantitative PCR detection method for human mitochondrial DNA copy number detection |
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2014
- 2014-10-20 CN CN201410558839.7A patent/CN104372002A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| JOE¨ LLE VERMEULEN ET AL: "Measurable impact of RNA quality on gene expression results from quantitative PCR", 《NUCLEIC ACIDS RESEARCH》 * |
| LIESBETH VOSSAERT ET AL: "Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells", 《BMC MOLECULAR BIOLOGY》 * |
| VICTOR DAYAN ET AL: "Human mesenchymal stromal cells improve scar thickness withoutv", 《INTERACTIVE CARDIOVASCULAR AND THORACIC SURGERY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116479105A (en) * | 2023-02-22 | 2023-07-25 | 中国疾病预防控制中心职业卫生与中毒控制所 | Nucleic acid reagent, kit and multiplex fluorescence quantitative PCR detection method for human mitochondrial DNA copy number detection |
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Effective date of registration: 20170309 Address after: 201499 Shanghai Road, Fengxian District, Lane 1698, Lane 17, building 26 Applicant after: Shanghai Yikang medical laboratory Co., Ltd. Applicant after: Taizhou Yikang Medical Inspection Co., Ltd. Address before: 5 building, TQB building, No.1, medicine City Avenue, Jiangsu, Taizhou, China Applicant before: YIKONGENOMICS. LTD |
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