CN104593515A - PCR-RFLP method for detecting H-site mutation of PIK3CA gene - Google Patents
PCR-RFLP method for detecting H-site mutation of PIK3CA gene Download PDFInfo
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Abstract
The invention discloses a PCR-RFLP method for detecting H1047R-site mutation of a PIK3CA gene. According to the method, a pair of specific primers are designed for the H1047R mutation site of the twentieth codon of the PIK3CA gene; and an Fst I digestion site is introduced to establish the PCR-RFLP method. The PCR-RFLP method mainly comprises the following steps: (1) extracting the genome DNA of a sample to be detected; (2) carrying out PCR amplification on a PIK3CA gene segment which contains the H1047R mutation site; (3) taking the PIK3CA gene segment as a template to carry out Fst I restriction enzyme reaction; (4) carrying out gel electrophoresis to obtain a length polymorphism atlas of restriction segments; and (5) determining the PIK3CA gene mutation state of the sample to be detected according to the length and quantity of the restriction fragments. The method has the advantages of being simple to operate, strong in specificity, high in sensitivity and low in cost, and can be used for effectively detecting the PIK3CA gene mutation state.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of PCR-RFLP method detecting PIK3CA gene H1047R site mutation.
Background technology
Phosphatidyl-inositol 3-kinase (PI3K) is the enzyme of 3 di making phosphatidylinositols molecule mysoinositol ring, and its signal path regulated and controled relates to the multiple vital movements such as cell proliferation, adhesion, existence, plays an important role in tumor development.The catalytic subunit of PIK3CA genes encoding PI3K, can activate the downstream AKT approach of PI3K path.The generation of the kinds of tumors such as the sudden change of PIK3CA and large bowel cancer, mammary cancer, lung cancer, ovarian cancer is closely related.Research finds, in large bowel cancer, the mutantional hotspot of PIK3CA concentrates on E542K, E545K and H1047R.Recent research shows, be positioned at the H1047R point mutation of extron 20 relevant to the targeted therapy curative effect of the anti-epidermal growth factor receptor EGFR of Metastatic Colorectal Cancer, the patient carrying this sudden change cannot benefit from anti-EGFR targeted therapy, PIK3CA H1047R suddenlys change likely after KRAS G12V suddenlys change and BRAF V600E suddenlys change, and becomes the another outcome prediction factor of large bowel cancer targeted therapy.Therefore, develop high efficiency gene mutation detection methods for this site for the applicable crowd of Accurate Prediction targeted therapy, improve result for the treatment of, the medical expense avoiding patient unnecessary and toxic side effect, carry out the treatment of individuation, significant.
At present, DNA direct Sequencing is the gold standard of PIK3CA detection in Gene Mutation, but the detection sensitivity of the method is lower, usually only has 20%-30%, is difficult to detect low-abundance transgenation in sample, easily causes the mis-classification of medication crowd, cause failing to respond to any medical treatment.In addition, DNA direct Sequencing method complex operation, consuming time.In recent years, there is research to set up the H1047R sudden change of HRM high-resolution fusion curve method detection PIK3CA, to a certain degree improve detection sensitivity, but higher testing cost and special equipment requirements limit its clinical application widely.
PCR-RFLP is called PCR-Sequence specific oligonucleotide probing, is a kind of detection method of gene mutation of maturation.Cut the distinguishing base site of wild-type and mutant DNA, electrophoretic separation endonuclease bamhi by restriction endonuclease specific recognition, according to the existence of the diversity judgement sudden change of fragment length and quantity, be particularly suitable for the detection to point mutation.Due to the method high specificity, result easily judges, simple to operate, is widely used.Relative to DNA direct Sequencing, the method has higher detection sensitivity; Relative to HRM etc. with the method for peak type diversity judgement result, the method has higher detection accuracy, and does not have complicated equipment requirements, and testing cost is relatively low, be applicable to clinical practical application, but have no the report adopting the method to carry out PIK3CA detection in Gene Mutation at present.For this reason, the present invention develops a kind of PCR-RFLP detection method for PIK3CA gene H1047R mutational site, is used to guide and monitors the individualized treatment of tumour.
Summary of the invention
The object of this invention is to provide a kind of detection method for PIK3CA gene H1047R site mutation.Invention main points are to design a pair Auele Specific Primer for PIK3CA gene H1047R mutational site, introduce Fst I restriction enzyme site, wild-type and saltant type PIKACA gene fragment is made to form otherness restriction map, analyze H1047R site mutation state, be used to guide tumor individual therapy.
The PCR-RFLP method that the present invention detects PIK3CA gene H1047R site mutation comprises the following steps:
1. extract the genomic dna of measuring samples
2. the pcr amplification of PIK3CA gene
With the genomic dna extracted for template, use upstream primer 5 '-GGAGTATTTCATGAAACAA ATGAATGATGCG-3 ' and downstream primer 5 '-GAGCTTTCATTTTCTCAGTTATCTT-3 ', carry out pcr amplification, obtain 126bp amplified production, introduce Fst I restriction enzyme site simultaneously;
3. Fst I endonuclease reaction
The 126bp product obtained to increase, for template, uses Fst I restriction endonuclease to carry out endonuclease reaction;
4. electrophoresis detection endonuclease bamhi
Adopt agarose gel electrophoresis method for detecting to be separated endonuclease bamhi, obtain restriction fragment length polymorphism collection of illustrative plates;
5. mutation status result judges
According to the length of restriction fragment and the PIK3CA transgenation state of quantity determination measuring samples, electrophoretogram presents two band persons and is judged to be PIK3CA gene wild-type in 96bp and 30bp position; Present a band person in electrophoretogram at 126bp place and be judged to be saltant type.
Described measuring samples comprises clone, flesh tissue, paraffin section, blood preparation or ight soil; The genomic dna of described measuring samples, the DNA extraction kit of commodity in use is extracted and is obtained.
Described PIK3CA gene PCR amplification system is as follows:
ddH
2O 17 μL
10 × buffer is (containing Mg
2+) 2.5 μ L
dNTP 2 μL
Upstream primer (10 μMs) 1 μ L
Downstream primer (10 μMs) 1 μ L
Genomic dna 1 μ L
rTaq 0.5 μL
Cumulative volume 25 μ L
Described pcr amplification condition is:
Denaturation: 94 DEG C of 5 min; 30 circulations: sex change 94 DEG C of 45 s, anneal 60 DEG C of 45 s, extends 72 DEG C of 45 s; Finally extend 72 DEG C of 5 min.
Described Fst I endonuclease reaction system and condition as follows:
ddH
2O 5 μL
10× FastDigest Buffer 1 μL
PCR product (200ng) 2 μ L
FastDigest enzyme（FstⅠ） 2 μL
Hatch 10 min for 37 DEG C.
Described electrophoresis detection endonuclease bamhi adopts agarose gel electrophoresis, and use 3% sepharose, 10 μ L digestion products loadings, 5 μ L DL500 contrast as molecular weight standard, carry out electrophoretic separation under 95V condition.EB dyes, and gel imaging system is observed and taken pictures.
Described mutation status result decision method is: electrophoretogram presents two band persons and is judged to be PIK3CA gene wild-type in 96bp and 30bp position, namely containing 1 Fst I restriction enzyme site; Present a band person in electrophoretogram at 126bp place and be judged to be saltant type, namely not containing Fst I restriction enzyme site.
Advantage of the present invention and beneficial effect as follows:
1. compared with DNA direct sequencing, simple to operate, amount of samples is little, highly sensitive.Using sepharose as electrophoresis matrix, EB dyeing display electrophoretic band, the present invention can reach 0.5% to the detection sensitivity of mutant DNA.Adopt the present invention to carry out the PIK3CA transgenation state-detection of 60 routine PATIENTS WITH LARGE BOWEL tumor tissues paraffin sections, sudden change recall rate is 20%, apparently higher than 10% of DNA direct sequencing.The present invention is as adopted polyacrylamide gel as matrix, and silver staining display electrophoretic band, its sensitivity can further improve.
2. the present invention cuts the existence of the specific fragment collection of illustrative plates judgement sudden change of generation according to enzyme, compared with the method adopting peak type judged result with HRM etc., have and better detect circulation ratio and accuracy, the present invention is adopted to detect the PIK3CA transgenation state of 60 routine PATIENTS WITH LARGE BOWEL tumor tissues paraffin sections, detect 12 examples and there is sudden change, its mutation status is all consistent with DNA direct Sequencing or cloning and sequencing detected result.
3., compared with the method such as DNA sequencing, HRM, quantitative fluorescent PCR, the present invention does not need complexity, expensive equipment, conveniently can complete detection in common Molecular Biology Lab.
4. detection sensitivity of the present invention is by being transplanted on the platforms such as micro-fluidic chip by electrophoresis link, is further improved.
5. can with the PCR-RFLP method combined utilization for KRAS, BRAF gene point mutation reported, multiple genes of being correlated with personalized treatment are detected under identical, higher detection sensitivity simultaneously, improves the accuracy of personalized medicine.
6. the present invention is applicable to the analysis of multiple detected object, comprises clone, flesh tissue sample, paraffin section, blood, ight soil etc.
Accompanying drawing explanation
Fig. 1 is that PCR-RFLP detects PIK3CA gene H1047R site mutation state electrophorogram in colorectal cancer cells.
Fig. 2 is that PCR-RFLP detects PIK3CA gene H1047R site mutation state electrophorogram in PATIENTS WITH LARGE BOWEL tumor tissues paraffin section sample.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1
PCR-RFLP method of the present invention detects PIK3CA gene H1047R site mutation in colorectal cancer cells and comprises the steps:
(1) genomic dna of sample is extracted
Get Human Large Intestine Carcinoma Cells system SW620 and LS174T being in logarithmic phase respectively, after tryptic digestion, counting cells.Get cell 1 × 10
6individual, centrifugal 5 min of 1000 rpm, abandon supernatant; The operation steps extracting test kit according to Promega full-length genome extracts cell genomic dna; The DNA extracted dissolves in 20 μ L ddH
2o, measuring under 260 nm wavelength and adjusting concentration is 0.5 μ g/ μ L;
(2) pcr amplification of PIK3CA gene
With genomic dna about 100 ng extracted for template, use upstream primer 5 '-GGAGTATTTCATGAAACAAATGAATGATGCG-3 ' and downstream primer 5 '-GAGCTTTCATTTTCTCAGTTATCTT-3 ', carry out pcr amplification, obtain 126bp amplified production;
It is 25 μ L that PCR reacts cumulative volume, and moiety is:
ddH
2O 17 μL
10 × buffer is (containing Mg
2+) 2.5 μ L
dNTP 2 μL
Upstream primer (10 μMs) 1 μ L
Downstream primer (10 μMs) 1 μ L
Genomic dna 1 μ L
rTaq 0.5 μL
Pcr amplification condition is: 94 DEG C of 5 min denaturation, then 94 DEG C of 45 s, 60 DEG C of 45 s, 72 DEG C of 45 s, totally 30 circulations, last 72 DEG C of 5 min;
(3) Fst I endonuclease reaction
Get pcr amplification product 2 μ L as template, carry out endonuclease reaction with Fst I restriction endonuclease;
Endonuclease reaction volume 10 μ L, system composition and enzyme tangent condition are:
ddH
2O 5 μL
10× FastDigest Buffer 1 μL
PCR product (200ng) 2 μ L
FastDigest enzyme（FstⅠ） 2 μL
Hatch 10 min for 37 DEG C;
(4) electrophoresis detection endonuclease bamhi
Prepare 3% sepharose, get 5 μ L DL500 Marker respectively, 10 μ L digestion products loadings, carry out electrophoretic separation under 95V condition.EB dyes, and gel imaging system is observed and taken pictures;
(5) mutation status result judges
As shown in Figure 1, the 1st swimming lane: DL500 DNA Marker, the 2nd swimming lane: SW620 cell, the 3rd swimming lane: LS174T cell.Shown by Fig. 1 result, the restriction enzyme digestion and electrophoresis collection of illustrative plates of SW620 cell presents two bands in 96bp and 30bp position, prompts for PIK3CA gene wild-type.LS174T cell electrophoresis collection of illustrative plates also presents two bands in 96bp and 30bp position, and prompting exists wild-type fragment; But also present a band at 126bp place simultaneously, prompt for saltant type, therefore infer that LS174T is the heterozygous mutant in PIK3CA gene H1047R site.The cell mutation state determined by this collection of illustrative plates is consistent with sequencing result, illustrates that the method that the present invention sets up accurately can detect PIK3CA gene H1047R site mutation.
Embodiment 2
PCR-RFLP method of the present invention detects PIK3CA gene H1047R site mutation in PATIENTS WITH LARGE BOWEL tumor tissues paraffin section sample and comprises the steps:
(1) genomic dna of sample is extracted
Get PATIENTS WITH LARGE BOWEL tumor tissues paraffin section (5 μm of thickness), the operation steps according to OMEGA paraffin section DNA extraction kit extracts the genomic dna of cell, final with 40 μ L elution DNA, for subsequent use;
(2) pcr amplification of PIK3CA gene
Getting DNA elutriant 2 μ L is template, uses upstream primer 5 '-GGAGTATTTCATGAAACAA ATGAATGATGCG-3 ' and downstream primer 5 '-GAGCTTTCATTTTCTCAGTTATCTT-3 ', carries out pcr amplification, obtain 126bp amplified production;
It is 25 μ L that PCR reacts cumulative volume, and moiety is:
ddH
2O 16 μL
10 × buffer is (containing Mg
2+) 2.5 μ L
dNTP 2 μL
Upstream primer (10 μMs) 1 μ L
Downstream primer (10 μMs) 1 μ L
Genomic dna 2 μ L
rTaq 0.5 μL
Pcr amplification condition is: 94 DEG C of 5 min denaturation, then 94 DEG C of 45 s, 60 DEG C of 45 s, 72 DEG C of 45 s, totally 30 circulations, last 72 DEG C of 5 min;
(3) Fst I endonuclease reaction
Get pcr amplification product 2 μ L as template, carry out endonuclease reaction with Fst I restriction endonuclease.
Endonuclease reaction volume 10 μ L, system composition and enzyme tangent condition are:
ddH
2O 5 μL
10× FastDigest Buffer 1 μL
PCR product (200ng) 2 μ L
FastDigest enzyme（FstⅠ） 2 μL
Hatch 10 min for 37 DEG C;
(4) electrophoresis detection endonuclease bamhi
Prepare 3% sepharose, get 5 μ L DL500 Marker respectively, 10 μ L digestion products loadings, carry out electrophoretic separation under 95V condition.EB dyes, and gel imaging system is observed and taken pictures.
(5) mutation status result judges
Restriction enzyme digestion and electrophoresis collection of illustrative plates presents two band persons and is judged as PIK3CA gene wild-type in 96bp and 30bp position; Person is judged as PIK3CA genic mutation type to there is band at 126bp place.As shown in Figure 2, M swimming lane: DL500 DNA Marker, 1-4 swimming lane: PATIENTS WITH LARGE BOWEL tumor tissues paraffin section sample 1-4.Shown by Fig. 2 result, sample 1 and 3 presents two bands in 96bp and 30bp position, prompts for PIK3CA gene wild-type; Except presenting except two bands in 96bp and 30bp position, also there is band at 126bp place in sample 2 and 4, illustrates containing saltant type PIK3CA gene.This result is consistent with DNA sequencing result, illustrates that method that the present invention sets up accurately can detect the PIK3CA gene H1047R site mutation state in tumor tissues paraffin section sample.
Claims (6)
1. detect a PCR-RFLP method for PIK3CA gene H1047R site mutation, it is characterized in that comprising following steps:
(1) genomic dna of measuring samples is extracted
(2) pcr amplification of PIK3CA gene
With the genomic dna extracted for template, use upstream primer 5 '-GGAGTATTTCATGAAACAA ATGAATGATGCG-3 ' and downstream primer 5 '-GAGCTTTCATTTTCTCAGTTATCTT-3 ', carry out pcr amplification, obtain 126bp amplified production, introduce Fst I restriction enzyme site simultaneously;
(3) Fst I endonuclease reaction
The 126bp product obtained to increase, for template, uses Fst I restriction endonuclease to carry out endonuclease reaction;
(4) electrophoresis detection endonuclease bamhi
Adopt agarose gel electrophoresis method for detecting to be separated endonuclease bamhi, obtain restriction fragment length polymorphism collection of illustrative plates;
(5) mutation status result judges
According to the length of restriction fragment and the PIK3CA transgenation state of quantity determination measuring samples, electrophoretogram presents two band persons and is judged to be PIK3CA gene wild-type in 96bp and 30bp position; Present a band person in electrophoretogram at 126bp place and be judged to be saltant type.
2. the PCR-RFLP method of detection PIK3CA gene H1047R site mutation according to claim 1, is characterized in that described measuring samples comprises clone, flesh tissue, paraffin section, blood preparation or ight soil; The genomic dna of described measuring samples, the DNA extraction kit of commodity in use is extracted and is obtained.
3. the PCR-RFLP method of detection PIK3CA gene H1047R site mutation according to claim 1, is characterized in that the PCR amplification system of described PIK3CA gene is as follows:
ddH
2O 17 μL
10 × buffer is (containing Mg
2+) 2.5 μ L
dNTP 2 μL
Upstream primer (10 μMs) 1 μ L
Downstream primer (10 μMs) 1 μ L
Genomic dna 1 μ L
rTaq 0.5 μL
Cumulative volume 25 μ L
Described pcr amplification condition is:
Denaturation: 94 DEG C of 5 min; 30 circulations: sex change 94 DEG C of 45 s, anneal 60 DEG C of 45 s, extends 72 DEG C of 45 s; Finally extend 72 DEG C of 5 min.
4. the PCR-RFLP method of detection PIK3CA gene H1047R site mutation according to claim 1, it is characterized in that described Fst I endonuclease reaction system and condition as follows:
ddH
2O 5 μL
10× FastDigest Buffer 1 μL
PCR product (200ng) 2 μ L
FastDigest enzyme（FstⅠ） 2 μL
Hatch 10 min for 37 DEG C.
5. the PCR-RFLP method of detection PIK3CA gene H1047R site mutation according to claim 1, it is characterized in that described electrophoresis detection endonuclease bamhi adopts agarose gel electrophoresis, use 3% sepharose, 10 μ L digestion products loadings, 5 μ L DL500 contrast as molecular weight standard, carry out electrophoretic separation under 95V condition, EB dyes, and gel imaging system is observed and taken pictures.
6. the PCR-RFLP method of detection PIK3CA gene H1047R site mutation according to claim 1, is characterized in that described mutation status result decision method is: electrophoretogram presents two band persons and is judged to be PIK3CA gene wild-type namely containing 1 Fst I restriction enzyme site in 96bp and 30bp position; Present a band person in electrophoretogram at 126bp place and be judged to be saltant type namely not containing Fst I restriction enzyme site.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858432A (en) * | 2017-12-11 | 2018-03-30 | 冷希岗 | A kind of kit and its application that PIK3CA mutation are detected by digital pcr |
Citations (4)
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|---|---|---|---|---|
| WO2008151031A1 (en) * | 2007-05-30 | 2008-12-11 | The Trustees Of Columbia University In The City Of New York | Detecting mutated gene sequences by mutant-enriched sequencing |
| WO2011087928A1 (en) * | 2010-01-12 | 2011-07-21 | Siemens Healthcare Diagnostics Inc. | Oligonucleotides and methods for detecting kras and pik3ca mutations |
| CN102234685A (en) * | 2010-04-23 | 2011-11-09 | 广州益善生物技术有限公司 | Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation |
| CN102533958A (en) * | 2010-12-28 | 2012-07-04 | 苏州科贝生物技术有限公司 | Method and kit for detecting human PIK3CA gene mutation |
-
2015
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008151031A1 (en) * | 2007-05-30 | 2008-12-11 | The Trustees Of Columbia University In The City Of New York | Detecting mutated gene sequences by mutant-enriched sequencing |
| WO2011087928A1 (en) * | 2010-01-12 | 2011-07-21 | Siemens Healthcare Diagnostics Inc. | Oligonucleotides and methods for detecting kras and pik3ca mutations |
| CN102234685A (en) * | 2010-04-23 | 2011-11-09 | 广州益善生物技术有限公司 | Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation |
| CN102533958A (en) * | 2010-12-28 | 2012-07-04 | 苏州科贝生物技术有限公司 | Method and kit for detecting human PIK3CA gene mutation |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858432A (en) * | 2017-12-11 | 2018-03-30 | 冷希岗 | A kind of kit and its application that PIK3CA mutation are detected by digital pcr |
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Inventor after: Fang Jin Inventor after: Li Wanming Inventor after: Hu Tingting Inventor after: Feng Yiming Inventor before: Fang Jin Inventor before: Li Wanming Inventor before: Zhou Tingting Inventor before: Feng Yiming |
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