CN108179182A - The film preparation method of poor point mutation a kind ofly - Google Patents
The film preparation method of poor point mutation a kind ofly Download PDFInfo
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- CN108179182A CN108179182A CN201810089815.XA CN201810089815A CN108179182A CN 108179182 A CN108179182 A CN 108179182A CN 201810089815 A CN201810089815 A CN 201810089815A CN 108179182 A CN108179182 A CN 108179182A
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- centrifuge tube
- hybridization
- point mutation
- pcr product
- film
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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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to film items to hybridize display technology field, discloses the film preparation method of poor point mutation a kind ofly, and PCR product is placed in 98 DEG C of PCR amplification instrument is denaturalized 10 minutes；Amplification pipe after denaturation is immediately inserted into the placement of ice bath box after taking out；Meanwhile the centrifuge tube equipped with A liquid is placed in boiling water bath heating；Centrifuge tube is taken out, PCR product is added in into the centrifuge tube；The centrifuge tube is placed in the hybridization of constant temperature hybridization instrument.Inventive film item hybridization point colour developing is clear, is as a result easy to interpretation；The present invention improves result interpretation efficiency and accuracy is high without increasing medical department cost.
Description
Technical field
The invention belongs to film item hybridization display technology field more particularly to the film preparation methods of a kind of poor point mutation.
Background technology
At present, the prior art commonly used in the trade is such：
Since poor Genotyping is carried out in Kunming, α poor point mutation film item result show it is unintelligible, film item it is miscellaneous
Intersection point color is light, seriously affects sentence read result.The reason of causing color light, be thought of as may be because Yunnan height above sea level compared with
Height, required temperature (98 DEG C) is not achieved in boiling water bath heat denatured in experimentation, causes DNA denaturation insufficient.
In the prior art, PCR product is directly added into the 15ml centrifuge tubes equipped with A liquid；
This centrifuge tube is placed in boiling water bath and is heated 10 minutes；
Centrifuge tube is taken out, this centrifuge tube is placed in the hybridization of constant temperature hybridization instrument.
In the prior art, non-deletion type gene of alpha thalassemia generally use PCR-DNA reverse dot blot hybridizations method carries out
Detection.
PCR polymerase chain amplifications：It designs special PCR primer and 5 ' ends is marked with biotin, amplification obtains non-
Deletion form α-thalassemia target gene fragment, the segment contain genotype mutations site to be detected.
DNA reverse dot blot hybridizations (reverrsedotblot, RDB)：According to detection site base difference, according to base complementrity
Pair principle designs specific oligonucleotide probe, for use probe is put on the specific position of nylon membrane, then will be to be measured respectively
DNA sample hybridize therewith, by corresponding chromogenic reaction with regard to hybridization signal can be showed.Therefore judge the big of a certain gene locus
Part or all of allele, for non-deletion type gene of alpha thalassemia parting.
In conclusion problem of the existing technology is：
(1) the film DNA denaturation of poor point mutation existingly is insufficient；Film item hybridization point colour developing is unintelligible, is as a result not easy
In interpretation；Accuracy is low；For height above sea level, the area of low boiling point, conventional experimental implementation can not meet requirement of experiment.
(2) it provides to walk using sub- non-deletion type gene of alpha thalassemia detection kit that can be biological, by the prior art
Rapid to carry out, the final experimental result of multiple batches of experiment all shows colour developing deficiency, it is difficult to judging result.
Solve the difficulty and meaning of above-mentioned technical problem：The present invention hybridizes point display clearly for film item, does not influence result
Interpretation is improved former experimental implementation.Good good effect is obtained.
Search experimental result it is undesirable during, the present invention consider during DNA reverse dot blot hybridizations, into
Before the hybridization of row DNA molecular, hybridization reaction pipe is placed in boiling water bath 10 minutes, by the method for heating, by double chain DNA molecule
It is separated into single-stranded, this process referred to as denaturation.Kunming is located in plateau, and the boiling point of boiling water is relatively low, if causes double chain DNA molecule
Unwinding is insufficient, and the kit of commercialization is in face of the area distribution of varying environment, and whether the difference in varying environment area is to experiment
There are Different Effects.For this purpose, the present invention improves.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of the film preparation method of poor point mutation.
The invention is realized in this way the film item of the film preparation method of poor point mutation, describedly poor point mutation a kind ofly
Preparation method includes：
PCR product is placed in 98 DEG C of PCR amplification instrument to be denaturalized 10 minutes；
Amplification pipe after denaturation is immediately inserted into the placement of ice bath box after taking out；
Meanwhile the centrifuge tube equipped with PCR product is placed in boiling water bath heating；
Centrifuge tube is taken out, PCR product is added in into the centrifuge tube；
The centrifuge tube is placed in the hybridization of constant temperature hybridization instrument.
Further, the 15ml centrifuge tubes equipped with PCR product are placed in boiling water bath to heat 10 minutes.
In conclusion advantages of the present invention and good effect are：
Inventive film item hybridization point colour developing is clear, is as a result easy to interpretation.
The present invention improves result interpretation efficiency and accuracy is high without increasing medical department cost.
Description of the drawings
Fig. 1 is the film preparation method flow chart of poor point mutation provided in an embodiment of the present inventionly.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Yunnan height above sea level, low boiling point, so conventional experimental implementation can not meet the requirement of experiment of Yunnan Province.
Fig. 1 is the film preparation method of poor point mutation provided in an embodiment of the present inventionly, including：
S101：PCR product is placed in 98 DEG C of PCR amplification instrument to be denaturalized 10 minutes；
S102：Amplification pipe after denaturation is immediately inserted into the placement of ice bath box after taking out；
S103：Meanwhile the 15ml centrifuge tubes equipped with PCR product are placed in boiling water bath and are heated 10 minutes；
S104：Centrifuge tube is taken out, PCR product is added in into the centrifuge tube；
S105：This centrifuge tube is placed in the hybridization of constant temperature hybridization instrument.
Inventive film item hybridization point colour developing is clear, is as a result easy to interpretation.
The present invention improves result interpretation efficiency and accuracy is high without increasing medical department cost.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (2)
1. the film preparation method of poor point mutation a kind ofly, which is characterized in that the film bar preparation method packet of poor point mutation describedly
It includes：
PCR product is placed in 98 DEG C of PCR amplification instrument to be denaturalized 10 minutes；
Amplification pipe after denaturation is immediately inserted into the placement of ice bath box after taking out；
Meanwhile the centrifuge tube equipped with PCR product is placed in boiling water bath heating, and hybridized.
2. the film preparation method of poor point mutation as described in claim 1, which is characterized in that
15ml centrifuge tubes equipped with PCR product are placed in boiling water bath heating hybridization 10 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810089815.XA CN108179182A (en) | 2018-01-30 | 2018-01-30 | The film preparation method of poor point mutation a kind ofly |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810089815.XA CN108179182A (en) | 2018-01-30 | 2018-01-30 | The film preparation method of poor point mutation a kind ofly |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108179182A true CN108179182A (en) | 2018-06-19 |
Family
ID=62551806
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810089815.XA Pending CN108179182A (en) | 2018-01-30 | 2018-01-30 | The film preparation method of poor point mutation a kind ofly |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108179182A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020064778A1 (en) * | 1998-08-13 | 2002-05-30 | Yu-Wen Hu | Primer-specific and mispair extension assay for identifying gene variation |
| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
-
2018
- 2018-01-30 CN CN201810089815.XA patent/CN108179182A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020064778A1 (en) * | 1998-08-13 | 2002-05-30 | Yu-Wen Hu | Primer-specific and mispair extension assay for identifying gene variation |
| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
Non-Patent Citations (4)
| Title |
|---|
| HELENE PUEHRINGER等: "Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions", 《CLIN CHEM LAB MED》 * |
| 张申等: "《分子生物学检验技术》", 28 February 2014, 华中科技大学出版社 * |
| 蔡望伟等: "《生物化学与分子生物学实验》", 31 August 2015, 华中科技大学出版社 * |
| 黄海龙等: "4种罕见β-地中海贫血基因检测膜条的制备及其临床应用", 《中国妇幼保健》 * |
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| PB01 | Publication | ||
| PB01 | Publication | ||
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Application publication date: 20180619 |
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| RJ01 | Rejection of invention patent application after publication |