CN103243113A - DNA (Deoxyribonucleic Acid) molecule, primer group for amplifying DNA molecule, kit comprising primer group and using method thereof - Google Patents
DNA (Deoxyribonucleic Acid) molecule, primer group for amplifying DNA molecule, kit comprising primer group and using method thereof Download PDFInfo
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- CN103243113A CN103243113A CN2013101859234A CN201310185923A CN103243113A CN 103243113 A CN103243113 A CN 103243113A CN 2013101859234 A CN2013101859234 A CN 2013101859234A CN 201310185923 A CN201310185923 A CN 201310185923A CN 103243113 A CN103243113 A CN 103243113A
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Abstract
The invention relates to the technical field of medicines and in particular relates to a DNA (Deoxyribonucleic Acid) molecule, a primer group for amplifying the DNA molecule, a kit comprising the primer group and a using method thereof. The provided DNA molecule is a mutant gene of human DDX11. By adopting the provided primer group, the fragments comprising the mutational site can be amplified through a polymerase chain reaction (PCR) mode, and whether the sample to be tested carries the mutant gene can be judged according to the sequencing result of the amplification product, so that whether a patient has the risk of suffering from cervical cancer is predicted, and early diagnosis of the cervical cancer is realized. The provided kit comprises a primer group which can be used for detecting whether the sample carries the mutational site on the human DDX11. Because the change of tissue cell morphology is caused by gene mutation, the primer is used for early diagnosis of the cervical cancer, and compared with the traditional technology, the primer has high sensitivity and high accuracy.
Description
Technical field
The present invention relates to medical technical field, relate in particular to dna molecular, the primer sets of this dna molecular that increases, the test kit that comprises this primer sets and using method thereof.
Background technology
Cervical cancer is the 3rd common malignancy that is only second to mammary cancer and colorectal cancer among the global women, is to be only second to mammary cancer to occupy the 2nd common malignancy in developing country, is modal female genital tract malignant tumour.Global New Development cases of cervical cancer 52.98 ten thousand in 2008, death 25.51 ten thousand people, wherein 85% new cases are in developing country.
The weight of the clinical symptom of cervical cancer is sooner or later relevant with the state of an illness, and early invasive carcinoma is generally asymptomatic under cervical intraepithelial neoplasia change and the mirror, how to find in generaI investigation.The symptom that Ib phase and later each phase occur the earliest mainly contains vaginal hemorrhage and vagina discharge opeing.If the cancer knurl is invaded the pelvic cavity reticular tissue, compressing bladder, rectum and sciatic nerve and influence lymph and during venous return frequent micturition, urgent urination, rectal tenesmus, constipation, lower abdominal pain, sciatica, lower limb can occur and swell and ache etc.Nephrohydrosis, uremia can appear in the compressing of cancer knurl or infringement ureter.Cachexy often occur because of long-term consumption whole latter stage.
The effect that cervical cancer is found ahead of time and treatment just has.The survival rate in 5 years of cervical cancer of first phase can reach 93%, the second phase is approximately 80%, if but to three phases and the fourth phase, the particularly fourth phase just have only about 20%.The patient of first phase or second phase can perform an operation, and can arrive three after dates and can only adopt radiotherapy or chemicotherapy, and the first phase patient that performs an operation can have 90% curative ratio, some in addition reach 98%.As seen, the early treatment of cervical cancer depends on early diagnosis.But early stage cervical cancer is often asymptomatic, and sign is also not obvious, makes a definite diagnosis and need carry out the diagnosis of three ladders.The first step, the cervical cytology inspection, i.e. vaginal exfoliated cell smear inspection, this is the main method of screening at present and early discovery cervical cancer.Second step was carried out vaginoscopy, and naked eyes are not seen obvious cancer kitchen range person to cervical smear cytology is suspicious or positive, and vaginoscope can amplify pathology 6～40 times, the trickle metamorphosis of direct viewing epithelium of cervix uteri and blood vessel under intense light source.The 3rd step was uterine neck and neck tube examination of living tissue, this is the most reliable and indispensable method of making a definite diagnosis precancerous lesions of uterine cervix and cervical cancer, when suspicious or the positive and biopsy of cervical smear cytolgical examination is negative, should scratch and scrape the cervical canal censorship, find adenocarcinoma cell as cervical smear, should go the fractional curettage art, be from uterine endometrium or cervical canal with clear and definite gland cancer.As seen, existing clinical diagnosis relies on morphologic change on traditional pathological section.Cervical cancer generally has been infiltrating carcinoma after classical symptom and sign occurring.And early cervical carcinoma often is difficult to distinguish with healthy tissues, and sign is also not obvious, causes very big difficulty to clinical diagnosis.Therefore, be badly in need of research and be applied to the early stage new diagnostic method of cervical cancer.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of dna molecular, the primer sets of this dna molecular that increases, the test kit that comprises this primer sets and using method thereof, and dna molecular provided by the invention is the mutator gene of human DDX11.Primer sets provided by the invention, can be used for increasing comprises the fragment of catastrophe point, according to can judging to the sequencing result of amplified production whether testing sample carries sudden change, thereby whether the prediction patient has the risk of suffering from cervical cancer, realizes the early diagnosis of cervical cancer.
Dna molecular provided by the invention is for being that the 1102nd Nucleotide and the 2183rd Nucleotide of the nucleotide sequence of NM_004399 is undergone mutation in the Genbank accession number, and its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
The Genbank accession number is the nucleotide sequence of NM_004399, is the gene order of normal human subject DDX11.DDX11 is a kind of helicase in the human body, and English name is: Homo sapiens DEAD/H (Asp-Glu-Ala-Asp/His) box helicase11, be called for short DDX11.The present invention checks order by the cervical cancer sample being carried out full genome exon, and utilizes cancer beside organism in contrast, finds that the DDX11 gene is undergone mutation in the cervical cancer sample.The site that DDX11 gene in the cervical cancer sample is undergone mutation has two, and the 1102nd Nucleotide sports A by G, and the 2183rd Nucleotide sports A by G.The sudden change of Nucleotide can make codon change, but because there is degeneracy in codon, the sudden change that is not all Nucleotide all is nonsynonymous mutation, and two site mutation in the dna molecular nucleotide sequence provided by the invention are all nonsynonymous mutation, have caused the variation of aminoacid sequence.The wherein sudden change of the 1102nd Nucleotide makes that corresponding amino acid sports Serine by proline(Pro) in the aminoacid sequence; The sudden change of the 2183rd Nucleotide makes that corresponding amino acid sports Histidine by arginine in the aminoacid sequence.
In the dna molecular provided by the invention, the dna molecular of nucleotide sequence is that the 1102nd Nucleotide sports the sequence that A obtains by G in the nucleotide sequence of NM_004399 for the Genbank accession number shown in SEQ ID NO:1; The dna molecular of nucleotide sequence is that the 2183rd Nucleotide sports the sequence that A obtains by G in the nucleotide sequence of NM_004399 for the Genbank accession number shown in SEQ ID NO:2; The dna molecular of nucleotide sequence is that the 1102nd Nucleotide sports A by G and the 2183rd Nucleotide sports the sequence that A obtains by G in the nucleotide sequence of NM_004399 for the Genbank accession number shown in SEQ ID NO:2.
In these two sites undergo mutation in arbitrary site, all might increase the risk of suffering from cervical cancer.Therefore, the invention provides the application of dna molecular provided by the invention in the cervical cancer diagnosis.
The invention provides the primer sets of amplification dna molecular provided by the invention, formed by the upstream primer with nucleotide sequence shown in the SEQ ID NO:4 and the downstream primer with nucleotide sequence shown in the SEQ ID NO:5.
The application of the primer sets that the present invention also provides upstream primer with nucleotide sequence shown in the SEQ ID NO:4 and had a downstream primer of nucleotide sequence shown in the SEQ ID NO:5 in detecting the 1102nd coding mutation of nucleotide sequence that the Genbank accession number is NM_004399.
The invention provides the primer sets of amplification dna molecular provided by the invention, formed by the upstream primer with nucleotide sequence shown in the SEQ ID NO:6 and the downstream primer with nucleotide sequence shown in the SEQ ID NO:7.
The application of the primer sets that the present invention also provides upstream primer with nucleotide sequence shown in the SEQ ID NO:6 and had a downstream primer of nucleotide sequence shown in the SEQ ID NO:7 in detecting the 2183rd coding mutation of nucleotide sequence that the Genbank accession number is NM_004399.
Adopt primer sets provided by the invention, by the mode of PCR, can increase obtains comprising the fragment of catastrophe point, according to judging to the sequencing result of amplified production whether testing sample carries sudden change, thereby whether the prediction patient has the risk of suffering from cervical cancer, realizes the early diagnosis of cervical cancer.Because the morphologic change of histocyte is because due to the transgenation, primer provided by the invention is used for the early diagnosis of cervical cancer to be compared with conventional art, has higher sensitivity and the accuracy of Geng Gao.
The invention provides a kind of test kit, comprising: first primer sets and second primer sets;
Wherein, the first primer sets middle and upper reaches primer has nucleotide sequence shown in the SEQ ID NO:2, and downstream primer has nucleotide sequence shown in the SEQ ID NO:3;
The second primer sets middle and upper reaches primer has nucleotide sequence shown in the SEQ ID NO:4, and downstream primer has nucleotide sequence shown in the SEQ ID NO:5.
As preferably, also comprise the Taq enzyme in the test kit provided by the invention.
The using method of test kit provided by the invention comprises the steps:
Step 1: extract the cervical tissue complete genome DNA;
Step 2: be template with the cervical tissue complete genome DNA, obtain first amplified production with the amplification of first primer sets, be that primer amplification obtains second amplified production with second primer sets, first, second amplified production is the fragment in the nucleotide sequence of NM_004399 for the Genbank accession number;
Step 3: get first and second amplified production through sequencing analysis, the Genbank accession number is that the 1102nd Nucleotide of nucleotide sequence of NM_004399 is that the Genbank accession number is that the 2183rd Nucleotide of nucleotide sequence of NM_004399 is A in A and/or second amplified production in first amplified production, then carries sudden change; The Genbank accession number is that the 1102nd Nucleotide of nucleotide sequence of NM_004399 is G in first amplified production, and the Genbank accession number is that the 2183rd Nucleotide of nucleotide sequence of NM_004399 is G in second amplified production, does not then carry sudden change.
Since the Genbank accession number be in the nucleotide sequence of NM_004399 in two catastrophe points on the 1102nd and the 2183rd arbitrary site undergo mutation, all might increase the risk of trouble cervical cancer.Therefore, the invention provides the application of dna molecular provided by the invention in the cervical cancer diagnosis.
In the using method of test kit provided by the invention, extraction cervical cancer sample complete genome DNA, amplification, order-checking step all can adopt ordinary method of the prior art.
Wherein Kuo Zeng program is:
The invention provides a kind of dna molecular, the primer sets of this dna molecular that increases, the test kit that comprises this primer sets and using method thereof, dna molecular provided by the invention is the mutator gene of human DDX11, empirical tests, the people who carries sudden change suffers from the risk increase of cervical cancer.Adopt primer sets provided by the invention, by the mode of PCR, can increase obtains comprising the fragment of catastrophe point, according to judging to the sequencing result of amplified production whether testing sample carries sudden change, thereby whether the prediction patient has the risk of suffering from cervical cancer, realizes the early diagnosis of cervical cancer.Test kit provided by the invention comprises primer sets provided by the invention, can be used for test sample and whether carries last two mutational sites that increase the ill risk of cervical cancer of human DDX11.Because the morphologic change of histocyte is because due to the transgenation, primer provided by the invention is used for the early diagnosis of cervical cancer to be compared with conventional art, has higher sensitivity and the accuracy of Geng Gao.
Description of drawings
Fig. 1 shows that first amplified production and second amplified production carry out the agarose gel electrophoresis result, and wherein swimming lane Maker shows the electrophoresis result of maker, and maker adopts the DL2 available from Takara company, 000DNA Marker; Swimming lane 1 shows the electrophoresis result of first amplified production; Swimming lane 2 shows the electrophoresis result of second amplified production.
Embodiment
The invention provides a kind of dna molecular, the primer sets of this dna molecular that increases, the test kit that comprises this primer sets and using method thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The reagent that the present invention adopts is all common commercially available product, all can buy in market.
Wherein, the test kit of extraction normal cell complete genome DNA employing is available from Qiagen's
Blood﹠amp; Tissue Kit; The enzyme that amplification is adopted is available from TaKaRa company
DNA Polymerase; Primer sets provided by the invention is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, and the step that checks order in the embodiment of the invention is finished by China big Gene science Services Co., Ltd.
Below in conjunction with embodiment, further set forth the present invention:
Get normal cell, adopt available from Qiagen's
Blood﹠amp; Tissue Kit test kit extracts the sample complete genome DNA, and operating process is carried out to specifications.
With the upstream primer with nucleotide sequence shown in the SEQ ID NO:4 and the downstream primer with nucleotide sequence shown in the SEQ ID NO:5 complete genome DNA is increased, obtain first amplified production; With the upstream primer with nucleotide sequence shown in the SEQ ID NO:6 and the downstream primer with nucleotide sequence shown in the SEQ ID NO:7 complete genome DNA is increased in addition, obtain second amplified production;
The Taq enzyme mixture that amplification is adopted is available from TaKaRa company
DNA Polymerase, amplification system is:
2×MightAmp Buffer Ver.2 12.5μL；
Upstream primer 0.5 μ L(10 μ M/ μ L);
Downstream primer 0.5 μ L(10 μ M/ μ L);
Complete genome DNA 50ng;
MightAmp DNA Polymerase 0.5μL（1.25U/50μL）；
DdH
2O supplies 25 μ L.
Amplification program is:
The PCR instrument that amplification is adopted is the Mygene Series Peltier Thermal Cycler available from the Hangzhou star, and model is MG96+.
First amplified production and second amplified production are carried out agarose gel electrophoresis, and electrophoresis result as shown in Figure 1; And first amplified production and second amplified production carried out sequencing analysis, order-checking is finished by handsome Bioisystech Co., Ltd.
Electrophoresis result shows that the clip size of first amplified production is 306bp, and the clip size of second amplified production is 415bp, and electrophoresis result meets expection.
Sequencing result is analyzed, find that two sites of this normal people all do not undergo mutation, show that two pairs of designed primers can be used for the purpose fragment that increases, the Taq enzyme that amplification is adopted has carried out the accurate amplification of high-fidelity to the purpose fragment, so our kit designed can be used for detecting the existing sudden change of DNA purpose fragment in the cervical cancer.
Claims (9)
1. dna molecular, it is characterized in that, for being that the 1102nd Nucleotide and the 2183rd Nucleotide of the nucleotide sequence of NM_004399 is undergone mutation in the Genbank accession number, its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
2. the application of dna molecular as claimed in claim 1 in the cervical cancer diagnosis.
3. the primer of the dna molecular according to claim 1 of increasing is right, it is characterized in that, is made up of the upstream primer with nucleotide sequence shown in the SEQ ID NO:4 and downstream primer with nucleotide sequence shown in the SEQ ID NO:5.
4. primer as claimed in claim 3 is to the application in detecting the 1102nd coding mutation of nucleotide sequence that the Genbank accession number is NM_004399.
5. the primer of the dna molecular according to claim 1 of increasing is right, it is characterized in that, is made up of the upstream primer with nucleotide sequence shown in the SEQ ID NO:6 and downstream primer with nucleotide sequence shown in the SEQ ID NO:7.
6. primer as claimed in claim 5 is to the application in detecting the 2183rd coding mutation of nucleotide sequence that the Genbank accession number is NM_004399.
7. a test kit is characterized in that, comprising: first primer is to right with second primer;
Wherein, the first primer sets middle and upper reaches primer has nucleotide sequence shown in the SEQ ID NO:4, and downstream primer has nucleotide sequence shown in the SEQ ID NO:5;
The second primer sets middle and upper reaches primer has nucleotide sequence shown in the SEQ ID NO:6, and downstream primer has nucleotide sequence shown in the SEQ ID NO:7.
8. test kit according to claim 7 is characterized in that, also comprises the Taq enzyme.
9. as the using method of test kit as described in claim 7 or 8, it is characterized in that, comprise the steps:
Step 1: extract the cervical tissue complete genome DNA;
Step 2: be template with the cervical tissue complete genome DNA, obtain first amplified production with described first primer sets amplification, be that primer amplification obtains second amplified production with described second primer sets, described first, second amplified production is the fragment in the nucleotide sequence of NM_004399 for the Genbank accession number;
Step 3: get first and second amplified production through sequencing analysis, the Genbank accession number is that the 1102nd Nucleotide of nucleotide sequence of NM_004399 is that the Genbank accession number is that the 2183rd Nucleotide of nucleotide sequence of NM_004399 is A in A and/or described second amplified production in described first amplified production, then carries sudden change; The Genbank accession number is that the 1102nd Nucleotide of nucleotide sequence of NM_004399 is G in described first amplified production, and the Genbank accession number is that the 2183rd Nucleotide of nucleotide sequence of NM_004399 is G in described second amplified production, does not then carry sudden change.
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| CN2013101859234A CN103243113A (en) | 2013-05-20 | 2013-05-20 | DNA (Deoxyribonucleic Acid) molecule, primer group for amplifying DNA molecule, kit comprising primer group and using method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546702A (en) * | 2018-04-10 | 2018-09-18 | 西安交通大学 | The siRNA for targeting long-chain non-coding RNA DDX11-AS1 and its application in liver cancer treatment |
| CN112813162A (en) * | 2021-01-05 | 2021-05-18 | 中山大学附属第五医院 | Application of DDX 19A-based method for promoting cervical squamous cell carcinoma metastasis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760529A (en) * | 2008-12-26 | 2010-06-30 | 上海基康生物技术有限公司 | Cervical cancer related locus detecting method |
| CN102925553A (en) * | 2012-09-07 | 2013-02-13 | 中山大学达安基因股份有限公司 | Fluorescence in situ hybridization detection kit for cervical carcinoma and preparation method thereof |
-
2013
- 2013-05-20 CN CN2013101859234A patent/CN103243113A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760529A (en) * | 2008-12-26 | 2010-06-30 | 上海基康生物技术有限公司 | Cervical cancer related locus detecting method |
| CN102925553A (en) * | 2012-09-07 | 2013-02-13 | 中山大学达安基因股份有限公司 | Fluorescence in situ hybridization detection kit for cervical carcinoma and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| CAPO-CHICHI,J.M,ET AL: "Homo sapiens DEAD/H(Asp-Glu-Ala-Asp/His)box helicase 11(DDX11),transcript variant 2,mRNA", 《NCBI GENBANK》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546702A (en) * | 2018-04-10 | 2018-09-18 | 西安交通大学 | The siRNA for targeting long-chain non-coding RNA DDX11-AS1 and its application in liver cancer treatment |
| CN108546702B (en) * | 2018-04-10 | 2021-03-12 | 西安交通大学 | siRNA of targeting long-chain non-coding RNA DDX11-AS1 and application thereof in liver cancer treatment |
| CN112813162A (en) * | 2021-01-05 | 2021-05-18 | 中山大学附属第五医院 | Application of DDX 19A-based method for promoting cervical squamous cell carcinoma metastasis |
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Application publication date: 20130814 |