CN103571947A - Kit for detecting hotspot mutation of KRAS gene and detection method thereof - Google Patents
Kit for detecting hotspot mutation of KRAS gene and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and discloses a kit for detecting the hotspot mutation of KRAS gene and a detection method thereof. The kit comprises a PNA (peptide nucleic acid) reagent; and the wild type KRAS gene sequence is closed by a clamp technology so as to improve the sensitivity of a sanger sequencing process and the detection rate of the KRAS gene mutation. The detection method disclosed by the invention is simple and safe to operate and does not need many expensive reagents, thereby being economical and efficient; and meanwhile, the detection method also has the advantage of short operation time, and the whole detection process is finished within 4-6 hours.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of test kit and detection method thereof for detection of the sudden change of KRAS gene hot.
Background technology
The gene that RAS gene family is relevant to human tumor has three kinds: HRAS, KRAS and NRAS, be positioned at respectively on 11,12 and No. 1 karyomit(e)s.KRAS because of coding 21kD ras albumen have another name called p21 gene.In RAS gene, KRAS has the greatest impact to human cancer, and it seems molecular switch: the path that can control regulating cell growth when normal; Occur when abnormal, cause cell to continue growth, and stop cell self-destructive.She participates in intracellular signal transmission, and when KRAS transgenation, this gene forever activates, and can not produce normal RAS albumen, makes Cellular Signaling Transduction Mediated disorderly, uncontrolled cellular proliferation and canceration.
KRAS gene can be standard state (being called wild-type) or error state (ERST) (saltant type).In normal physiological situation, while activating the signal paths such as somatomedin after cell is subject to external stimulus, the K-Ras of wild-type by Tyrosylprotein kinase phosphorylations such as active growth factor after of short duration activation, KRAS after activation can activate the downstream signal albumen in this signal path, then the rapid inactivation of K-ras.KRAS activation/inactivating effect is controlled.Saltant type K-Ras albumen causes protein function abnormal, and still in state of activation, its functional status is uncontrollable, leads oncogenic lasting propagation etc. under without growth factor activation token stimulus.
Method for detection in Gene Mutation has Sanger sequencing, high performance liquid chromatography, real-time fluorescence quantitative PCR method etc. at present.Sanger gene sequencing technology has been passed through the development of 30 years and perfect, can check order to reaching the DNA fragmentation of 1,000bp now, and each base read to accuracy rate up to 99.999%.Owing to having the very high accuracy rate that reads, the order-checking of Sanger method becomes the gold standard of the genetic analysiss such as transgenation, single nucleotide polymorphism.
In tumor tissues, KRAS wild-type cell and mutant cell mix, and the sensitivity of Sanger sequencing is only 10～20%, when mutant cell account for whole detection cell 10～20% time just can detect, therefore limited greatly the application of Sanger sequencing.
Peptide nucleic acid(PNA) (peptide nucleic acids, a PNA) class replaces the DNA analogue of sugared phosphate backbone with polypeptide backbone.It is on the basis of the first-generation, s-generation antisense reagent, by Computer Design, build the third generation antisense reagent of also final synthetic, it is a kind of brand-new DNA analogue, with neutral peptide chain acid amides 2-amino-ethyl glycine key, replaced the pentose phosphate diester linkage skeleton in DNA, remaining is identical with DNA, PNA can, by the form identification of Watson-Crick base pairing and in conjunction with DNA or RNA sequence, form stable double-spiral structure.Because PNA is not electronegative, and there is not electrostatic repulsion between DNA and RNA, thereby the stability of combination and all greatly raisings of specificity; Be different from the hybridization between DNA or DNA, RNA, system salt concn is hybridized in the hybridization of PNA and DNA or RNA hardly to be affected, be much better than DNA/DNA or DNA/RNA with the hybridization ability of DNA or RNA molecule, show very high hybridization stability, good distinguished sequence recognition capability, not by nuclease and protease hydrolysis.And when PNA and complementary DNA have 1 base and do not mate, its solvent temperature can decline 8 ℃～20 ℃, when 2 bases are not mated, can not hybridize completely.In view of the advantage that above-mentioned many DNA moleculars do not possess, nearly ten years, people have found purposes for it in many high-tech sectors.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of test kit and detection method thereof for detection of the sudden change of KRAS gene hot is provided, the present invention adopts PNA euglycemic clamp sealing wild-type BRAF gene order, thereby improves susceptibility and the sudden change recall rate that detects the sudden change of KRAS gene hot.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
A kind of test kit for detection of the sudden change of KRAS gene hot, comprise PCR reaction solution, PNA reagent, enzymic digestion liquid, sequencing reaction liquid, magnetic bead and product purification liquid, described PCR reaction solution comprises PCR damping fluid, dNTPs, PCR forward primer, PCR reverse primer, probe and archaeal dna polymerase, the sequence of described PCR forward primer is SEQ.ID NO.1, the sequence of described PCR reverse primer is SEQ.ID NO.2, the sequence of described probe is SEQ.ID NO.3, the fluorophor of 5 ' end of described probe is FAM, and the fluorophor of 3 ' end is BHQ1; The sequence of described PNA is SEQ.ID NO.4; In described sequencing reaction liquid, contain Bigdye, Bigdye damping fluid and sequencing primer.
In such scheme, described PCR damping fluid is by 100mmol/L Tris-HCl, 500mmol/L KCl and 15mmol/L MgCl
2form; The volumetric molar concentration of described dNTPs is 2.5mmol/L dNTPs; The volumetric molar concentration of described PCR forward primer is 10 μ mol/L, and the volumetric molar concentration of described PCR reverse primer is 10 μ mol/L; The volumetric molar concentration of described probe is 10 μ mol/L; The enzyme activity unit concentration of described archaeal dna polymerase is 5U/ μ l; The volumetric molar concentration of described PNA reagent is 10 μ mol/L.
In such scheme, described enzymic digestion liquid comprises exonuclease I and alkaline phosphatase.
In such scheme, the enzyme activity unit of described exonuclease I and alkaline phosphatase is than being 5:2.
In such scheme, the sequencing primer that described sequencing reaction liquid contains 5 * Bigdye, 2.5 * Bigdye damping fluid and 3 μ mol/L.
In such scheme, described product purification liquid is 0.75M sodium chloride solution.
In such scheme, at 5 ' of described PCR forward primer, hold and insert general M13 sequence:
TGTAAAACGACGGCCAGT, holds and inserts general M13 sequence at 5 ' of described PCR reverse primer:
CAGGAAACAGCTATGACC, described sequencing primer sequence is:
TGTAAAACGACGGCCAGT。
Utilize mentioned reagent box to detect a method for BRAF gene hot sudden change, specifically comprise the steps:
(1) goal gene fluorescent quantitative PCR: use fluorescence quantitative PCR reaction solution and PNA reagent in test kit, sample DNA is carried out to pcr amplification reaction, obtain quantitative result and the PCR product of goal gene;
(2) according to the quantitative result in step (1), use the enzymic digestion liquid in test kit, the PCR product that step (1) is obtained carries out purifying, obtains PCR product after purifying;
(3) use the sequencing reaction liquid in test kit, the PCR product PCR reaction of checking order after the purifying that step (2) is obtained, PCR product obtains checking order;
(4) use magnetic bead and the product purification liquid in test kit, the order-checking PCR product that step (3) is obtained carries out purifying, and PCR product obtains checking order after purifying;
(5) PCR product application of sample to the sequenator that checks order after purifying step (4) being obtained carries out sequential analysis, order-checking gained gene order and the NCBI(U.S. state-run biotechnology information center) KRAS gene standard sequence is compared in database, and check peak figure, judge whether to exist KRAS gene hot sudden change: if the cover peak that typically suddenlys change, 12 GGTFeng Tu downstreams, site, KRAS gene hot Sudden change region in peak figure, or be entirely sudden change peak figure, in known sample, partly or entirely cell contains 12 sites, KRAS hot spot mutation region.Beneficial effect of the present invention:
(1) test kit of the present invention comprises PNA reagent, and sample DNA is being carried out in pcr amplification reaction, utilizes PNA reagent sealing wild-type KRAS gene order, improves the sensitivity of sanger sequencing and the recall rate of KRAS transgenation.
(2) simple to operate, the safety of detection method of the present invention, does not need the use of more expensive reagent simultaneously, economical and efficient.
(3) the detection method operating time of the present invention short, whole testing process can complete in 4～6 hours.
Accompanying drawing explanation
Fig. 1 is KRAS transgenation hot spot region sequence, and wherein underscore represents primer sequence or primer binding site, and square frame represents probe sequence or probe binding site, and a lower stroke dotted line represents PNA sequence or PNA binding site, and tilted letter represents mutational site.
Fig. 2 is KRAS gene by fluorescence quantitative pcr amplification curve in embodiment 1.
Fig. 3 is KRAS gene sequencing figure in embodiment 1.
Embodiment
In order to understand better the present invention, below in conjunction with accompanying drawing, form, embodiment, further illustrate content of the present invention, but content of the present invention is not only confined to example below.
Embodiment 1
1, synthesizing of the primer in PCR reaction solution, probe sequence and synthesizing of PNA sequence in test kit
The (see figure 1) that requires according to KRAS gene hot Sudden change region and order-checking fragment, designs following primer, probe and PNA, and synthesizes following primer, probe and PNA by the method for synthetic:
KRAS12F：ACTGGTGGAGTATTTGATAGTGTA（SEQ.ID?NO.1）
KRAS12R：CTTTATCTGTATCAAAGAATGGTCCTG（SEQ.ID?NO.2）
KRAS12P：FAM-ACCTTATGTGTGACATGTTCTAATATAGT-BHQ1（SEQ.ID?NO.3）
KRAS12PNA：GGAGCTGGTGGCGTAGGC（SEQ.ID?NO.4）
Above-mentioned primer, probe and PNA sequence are 5 ' end to 3 ' end, and 12 represent KRAS mutantional hotspot area password numbering, and F represents forward primer, and R represents reverse primer, and P represents probe, and FAM represents fluorophor in probe, and BHQ1 represents quenching group.
5 ' the end at above-mentioned forward primer inserts general M13 sequence: TGTAAAACGACGGCCAGT, 5 ' the end at reverse primer inserts general M13 sequence: CAGGAAACAGCTATGACC, the meaning that this universal primer inserts be to make after this experimental program in sequencing reaction all use unified sequencing primer.
2, the fluorescent quantitative PCR of goal gene fragment: utilize above-mentioned synthetic PCR primer, probe and PNA sequence, react by fluorescent quantitative PCR, obtain quantitative fluorescent PCR product.
(1) extract sample DNA: synthetic KRAS transgenation hot spot region wild-type and mutated genes, the sequence of KRAS transgenation hot spot region wild-type is SEQ.ID NO.5, the sequence of KRAS transgenation hot spot region saltant type is SEQ.ID NO.6, after being transformed into respectively DH5 α intestinal bacteria, cultivate, then take wild-type bacteria: saltant type bacteria concentration is than being 99:1 mixing, extract again mixed bacteria liquid DNA, obtain sample DNA;
(2) use PCR reaction solution and PNA reagent in test kit, above-mentioned sample DNA is carried out to Fluorescence PCR amplification, obtain object sheet segment DNA, PCR reaction system is 25 μ l systems:
Above-mentioned PCR reaction solution contains 10 * 100mmol/L Tris-HCl, 500mmol/L KCl, 15mmol/LMgCl
2,, 2.5mmol/L dNTPs, 10 μ mol/L forward primers, 10 μ mol/L reverse primers, 10 μ mol/L probes, 5U/ μ l Taq archaeal dna polymerase; The volumetric molar concentration of above-mentioned PNA reagent is 10 μ mol/L; Wherein the sequence of forward primer is SEQ.ID NO.1, and the sequence of reverse primer is SEQ.ID NO.2, and the sequence of probe is SEQ.ID NO.3, and the sequence of PNA is SEQ.ID NO.4.
(3) pcr amplification program is as following table:
Table 1PCR amplification program
(4) obtain pcr amplification product and fluorescent quantitative PCR result curve, according to fluorescent PCR amplification curve (Fig. 3), judge having or not and specificity of pcr amplification product: as can be seen from Figure 3, pcr amplification curve becomes typical " S " type curve, line smoothing, without obvious background signal, this explanation has specific pcr amplification product.
3, the purifying of PCR product
Get above-mentioned PCR reaction product 5 μ l and be placed in 1 pipe, every pipe adds enzymic digestion liquid 3 μ l in test kit, mixes, and after moment is centrifugal, 37 ℃ are heated 45 minutes, and 80 ℃ are heated 15 minutes.This enzymic digestion liquid is comprised of 1U/ μ l alkaline phosphatase 2 μ l and 5U/ μ l exonuclease I 1 μ l, can remove PCR and react remaining primer, dNTP, single stranded DNA etc., thereby obtain PCR product after purifying.
4, order-checking PCR reaction
Use sequencing reaction liquid in test kit, get after the above-mentioned purifying obtaining the PCR product 3 μ l PCR reaction of checking order after the digestion of enzymic digestion liquid, PCR product obtains checking order.
Sequencing reaction system is as follows:
Sequencing reaction liquid 1.75 μ l
PCR product 3 μ l after purifying
Distilled water 1.25 μ l
The sequencing primer that above-mentioned sequencing reaction liquid contains 5 * Bigdye, 2.5 * Bigdye damping fluid and 3 μ mol/L, wherein the sequence of sequencing primer is: TGTAAAACGACGGCCAGT.
Sequencing reaction program is as following table 2:
Table 2 order-checking PCR response procedures
5, adopt paramagnetic particle method purifying order-checking PCR product
(1) get above-mentioned order-checking PCR product 6 μ l, add 54 μ l0.75M NaCl, add 30 μ l Virahols and 10 μ l magnetic beads, after mixing, standing 3 minutes of room temperature;
(2) be placed in the standing 1min of magnetic frame, abandon waste liquid;
(3) add 200 μ l70% ethanol again, mix, standing 1min, is placed in magnetic frame standing 1 minute;
(4) abandon waste liquid, liquid feed is blotted, room temperature is dried;
(5) then add 20 μ l distilled waters, mix, place 3min for 60 ℃, be placed in the standing 1min of magnetic frame, get supernatant 10 μ l, be placed in 95 ℃, 5min, then be placed on-20 ℃, 5min, PCR product obtains checking order after purifying.
6, order-checking PCR product application of sample to the sequenator after purifying checks order
PCR product application of sample to the sequenator that checks order after above-mentioned 10 μ l purifying is checked order, sequenator WeiABI company 3500 sequenators, order-checking pattern is mode standard or quick mode, obtain sequencing result and see Fig. 3, the state-run biotechnology of the gained gene order that checks order in Fig. 3 and NCBI(U.S. information center) in database, KRAS gene standard sequence is compared, and check peak figure, the cover peak that typically suddenlys change, 12 GGTFeng Tu downstreams, site, KRAS gene hot Sudden change region in peak figure, in known sample, to contain KRAS hot spot mutation region 12 site mutations be GTT to part cell.Peak figure and the sudden change situation in sample of above-mentioned order-checking gained are in full accord.
Obviously, above-described embodiment is to be only the example that clearly explanation is done, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of therefore amplifying or change are still within the protection domain in the invention.
Claims (8)
1. the test kit for detection of KRAS gene hot sudden change, it is characterized in that, comprise PCR reaction solution, PNA reagent, enzymic digestion liquid, sequencing reaction liquid, magnetic bead and product purification liquid, described PCR reaction solution comprises PCR damping fluid, dNTPs, PCR forward primer, PCR reverse primer, probe and archaeal dna polymerase, the sequence of described PCR forward primer is SEQ.ID NO.1, the sequence of described PCR reverse primer is SEQ.ID NO.2, the sequence of described probe is SEQ.ID NO.3, the fluorophor of 5 ' end of described probe is FAM, the fluorophor of 3 ' end is BHQ1, the sequence of described PNA is SEQ.ID NO.4, described sequencing reaction liquid comprises Bigdye, Bigdye damping fluid and sequencing primer.
2. test kit according to claim 1, is characterized in that, described PCR damping fluid is by 100mmol/LTris-HCl, 500mmol/L KCl and 15mmol/L MgCl
2form; The volumetric molar concentration of described dNTPs is 2.5mmol/L dNTPs; The volumetric molar concentration of described PCR forward primer is 10 μ mol/L, and the volumetric molar concentration of described PCR reverse primer is 10 μ mol/L; The volumetric molar concentration of described probe is 10 μ mol/L; The enzyme activity unit concentration of described archaeal dna polymerase is 5U/ μ l; The volumetric molar concentration of described PNA reagent is 10 μ mol/L.
3. test kit according to claim 1, is characterized in that described enzymic digestion liquid comprises exonuclease I and alkaline phosphatase.
4. test kit according to claim 3, is characterized in that the enzyme activity unit of described exonuclease I and alkaline phosphatase is than being 5:2.
5. test kit according to claim 1, is characterized in that the sequencing primer that described sequencing reaction liquid contains 5 * Bigdye, 2.5 * Bigdye damping fluid and 3 μ mol/L.
6. test kit according to claim 1, is characterized in that described product purification liquid is 0.75M sodium chloride solution.
7. test kit according to claim 1, is characterized in that the 5 ' end at described PCR forward primer
Insert general M13 sequence: TGTAAAACGACGGCCAGT, at 5 ' end insertion general M13 sequence: CAGGAAACAGCTATGACC of described PCR reverse primer, described sequencing primer sequence is: TGTAAAACGACGGCCAGT.
8. right to use requires test kit described in 1～7 to detect the method that KRAS gene hot is suddenlyd change, and it is characterized in that it comprises the steps:
(1) goal gene fluorescent quantitative PCR: use PCR reaction solution and PNA reagent in test kit, sample DNA is carried out to pcr amplification reaction, obtain fluorescent quantitation result and the PCR product of goal gene;
(2) according to the quantitative result in step (1), use the enzymic digestion liquid in test kit, the PCR product that step (1) is obtained carries out purifying, obtains PCR product after purifying;
(3) use the sequencing reaction liquid in test kit, the PCR product PCR reaction of checking order after the purifying that step (2) is obtained, PCR product obtains checking order;
(4) use magnetic bead and the product purification liquid in test kit, the order-checking PCR product that step (3) is obtained carries out purifying, and PCR product obtains checking order after purifying;
(5) PCR product application of sample to the sequenator that checks order after purifying step (4) being obtained carries out sequential analysis, and order-checking gained gene order and KRAS gene standard sequence are compared, and check peak figure, judges whether to exist the sudden change of KRAS gene hot.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498615A (en) * | 2015-01-06 | 2015-04-08 | 浙江诺辉生物技术有限公司 | Primer and probe for detecting mutant KRAS genes |
| CN104805208A (en) * | 2015-04-30 | 2015-07-29 | 山东维真生物科技有限公司 | Primer-probe composition, kit and detection method for detecting seven kinds of hot-spot mutation of KRAS gene of humans |
| CN104830981A (en) * | 2015-04-29 | 2015-08-12 | 广州和实生物技术有限公司 | KRAS gene multipoint mutation single tube rapid detection method and kit |
| CN110791555A (en) * | 2019-11-20 | 2020-02-14 | 依科赛生物科技（太仓）有限公司 | Single-stranded DNA quantitative detection kit and preparation method and application thereof |
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| WO2008037694A1 (en) * | 2006-09-26 | 2008-04-03 | Diasorin Spa | Method for detection of mutant alleles combining real time pcr and rems-pcr |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498615A (en) * | 2015-01-06 | 2015-04-08 | 浙江诺辉生物技术有限公司 | Primer and probe for detecting mutant KRAS genes |
| CN104830981A (en) * | 2015-04-29 | 2015-08-12 | 广州和实生物技术有限公司 | KRAS gene multipoint mutation single tube rapid detection method and kit |
| CN104805208A (en) * | 2015-04-30 | 2015-07-29 | 山东维真生物科技有限公司 | Primer-probe composition, kit and detection method for detecting seven kinds of hot-spot mutation of KRAS gene of humans |
| CN104805208B (en) * | 2015-04-30 | 2017-12-05 | 山东维真生物科技有限公司 | For detecting the primer combination of probe thing, kit and detection method of 7 kinds of hot spot mutations of mankind KRAS genes |
| CN110791555A (en) * | 2019-11-20 | 2020-02-14 | 依科赛生物科技（太仓）有限公司 | Single-stranded DNA quantitative detection kit and preparation method and application thereof |
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