CN107475442A - A kind of method of microsatellite instability detection - Google Patents

A kind of method of microsatellite instability detection Download PDF

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CN107475442A
CN107475442A CN201710961022.8A CN201710961022A CN107475442A CN 107475442 A CN107475442 A CN 107475442A CN 201710961022 A CN201710961022 A CN 201710961022A CN 107475442 A CN107475442 A CN 107475442A
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primer pair
primer
seq
colorectal cancer
concentration
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张云静
李威
张中娜
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Biological Engineering (shanghai) Ltd By Share Ltd
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Abstract

The invention discloses a kind of method of microsatellite instability detection, it is related to molecular diagnosis field.The method of colorectal cancer microsatellite instability detection disclosed by the invention is based on multiple fluorescence PCR technology and capillary electrophoresis technique,With the first primer pair shown in SEQ ID NO.1 2,The second primer pair shown in SEQ ID NO.3 4,Three-primer pair shown in SEQ ID NO.5 6,The 5th primer pair shown in the 4th primer pair and SEQ ID NO.9 10 shown in SEQ ID NO.7 8 carries out multiplex PCR and detects 5 microsatellite locus BAT 25 simultaneously,BAT‑26,D2S123,D5S346 and D17S250,With higher sensitivity and precision,And introduce internal reference microsatellite locus Penta C detection,For excluding the pollution of the exogenous DNA in sample to be checked,Improve the confidence level of result.

Description

A kind of method of microsatellite instability detection
Technical field
The present invention relates to molecular diagnosis field, in particular to a kind of method of microsatellite instability detection.
Background technology
Colorectal cancer (carcinoma of colon and rectum, CRC) is that a kind of common serious harm mankind are good for The malignant tumour of health.With the development of human society, the life style and living environment of people are changed, life stress day Benefit increase, the pollution getting worse of living environment, cause the incidence of disease of colorectal cancer in recent years that obvious ascendant trend is presented, and And rejuvenation situation is presented in age of onset.Heredity colorectal cancer accounts for important proportion in colorectal cancer system, and it has heredity Property, the feature such as frequently-occurring, multiple and tumorigenic multiple organ, clinically according to polyposis multiple is whether there is, by heredity Property colorectal cancer is divided into hereditary polyposis and the major class of hereditary nonpolyposis colorectal cancer (HNPCC) two, two class heredity Colorectal cancer is all autosomal dominant inheritance.It is reported that hereditary nonpolyposis colorectal cancer accounts for colorectal cancer 15% or so.
Colorectal cancer is divided into that chromosome instability is fixed and microsatellite instability two types from mechanism.Microsatellite is time STR in cloth human genome.Microsatellite instability (Microsatellite Instability, MSI) is Refer to and cause the DNA of the tumour microsatellite length hair with same individual normal embryonal system DNA compared with due to lacking or inserting recurring unit It is raw to change, that is, produce the allele of odd length.Research shows, 15% colorectal cancer is occurred through MSI approach, MSI be by Defect occurs in mismatch repair gene to cause, and hereditary nonpolyposis colorectal carcinoma (HNPCC) is by mismatch repair gene embryonal system Dominant hereditary disease caused by mutation, more than 90% typical HNPCC patient have MSI features.In addition, also have 15% or so dissipate Hair property colorectal cancer patients have MSI features.The main reason for MSI HNPCC occurs is the mispairing reparation of reproduction cell (mismatch repair, MMR) gene mutation.Any one or several mispairing such as hMLHl, hMSH2, hMSH6 and hPMS2 are repaiied The inactivation of multiple genes leads to MSI, distributes the most only mismatch repair gene somatic mutations of Patients with Colorectal Cancer.
1997, MSI international research cooperative association was detected in the microsatellite instability for tumour and replication error phenotype International conference formulated unified MSI examination criterias, i.e. Bethesda Guidelines.The ginseng of the clear and definite MSI detections of the standard Examine detection site, including the microsatellite locus (BAT-25 and BAT-26) of two mononucleotide repeat units and three dinucleotides The microsatellite locus (D2S123, D5S346 and D17S250) of repeat unit.Using the MSI states of this standard detection tumor sample When, if 5 microsatellite locus detect 2 or the site of more than 2 changes, it is referred to as MSI-H types, i.e. high frequency Microsatellite instability is spent, MSI-H type tumour tables select distinctive clinical and pathogenic phenotypes.If 5 microsatellite locus are only examined Measure a site to change, be then referred to as MSI-L types, i.e. low-frequency degree microsatellite instability.5 microsatellite locus all do not have Change, be then referred to as MSS types, i.e. microsatellite stable type.Generally, due to MSI-L types and MSS types clinical manifestation without obvious Difference, so MSI-L types are included in into MSS types.
MSI colorectal cancers have significant difference compared with MSS colorectal cancers, in prognosis and treatment etc., and MSI knots are directly Intestinal cancer detection clinical meaning includes prognostic, three aspects of predictive and HNPCC auxiliary diagnosis and examination.The MSI-H II phases tie The carcinoma of the rectum is higher than MSS II phase colorectal cancers in 5 years without recurrence survival rate.I.e. in II phase colorectal cancer patients, MSI-H states carry Show good prognosis.5 FU 5 fluorouracil is the important chemotherapeutics of post operative colo-rectal cancer auxiliary treatment.MSH-H II phase colorectal cancers Patient has good prognosis but can not benefited from 5 FU 5 fluorouracil treatment.The patient screened from MSI-H colorectal cancers, Individually early diagnosis and preventative polypectomy can reduce the CRC death rates.Further, it is also possible to for patients' family into Member provides tumor prevention and examination.So clinically using MSI as colorectal cancer prognosis and the weight of formulation supplemental treatment regimens Molecular marked compound is wanted, and assists HNPCC screening.In the National on colorectal cancer examination in 2011 In Comprehensive Cancer Network guides, MSI detections are included in necessary detection project.It is in May, 2017, beautiful Food and medicine Surveillance Authority of state (Food and Drug Administration, FDA) announces " to accelerate approval PD-1 for the first time Antibody is used to determine there is frequent the microsatellite instability either adult of mismatch repair gene defect and children's late period or transfer Property solid tumor patient ".Clear stipulaties only have the trouble of frequent microsatellite instability or mismatch repair gene defect in FDA certifications Person can just have higher probability to benefit from PD-1 (programmed death 1) antibody, and AUTHORITATIVE DATA shows to resist with PD-1 Before body treatment, MSI detections are first done, because only that MSI-H type patients, receive PD-1 Antybody therapies, it is efficient just higher, MSS type patients are efficient very low or even extremely low.
The MSI detection techniques of traditional PCR-based technology generally have two kinds, and one kind is by microsatellite locus to be checked The sequence in downstream enters performing PCR amplification, and amplified production polyacrylamide gel electrophoresis, then by various display systems, (radiation is certainly Development, silver staining etc.) analyze the change for whetheing there is mobility；Another technology is direct sequencing, i.e., direct to pcr amplification product Sanger is sequenced to detect insertion or the missing whether microsatellite locus occurs repeat unit.But above two detection technique It is all each defective.Technique of polyacrylamide gel electrophoresis exists cumbersome, and time-consuming, and the low grade low with sensitivity of flux much lacks Fall into.The defects of expensive, required experiment condition is higher, and flux is not high be present in direct sequencing.In addition, treated in genome It is low to examine the sequence copy numbers of microsatellite locus, it is difficult to directly be detected.In addition, according to five microsatellite locus, two lists to be checked Nucleotide repeating unit and three dinucleotides repeat units, the change of microsatellite length is smaller, microsatellite length change base This is less than 25bp, and the MSI detection methods sensitivity of traditional PCR-based method and accuracy be all than relatively low, and completion five Individual site primer need to carry out multiple PCR reactions and it is follow-up corresponding to experiment flow could complete the detection of a sample.So Develop a kind of high sensitivity, high accurancy and precision, high flux, a simple to operate and tubular type while the method gesture for completing five site primers It must go.
In consideration of it, special propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of method of colorectal cancer microsatellite instability detection, this method has Gao Ling Sensitivity, high accurancy and precision, high flux, it is simple to operate the features such as.
What the present invention was realized in：
A kind of method of colorectal cancer microsatellite instability detection, it includes：Enter performing PCR with primer pair measuring samples to expand Increase；
Wherein, the primer is included for the detection primer of microsatellite locus to be checked amplification and for internal reference microsatellite locus The internal control primer of amplification；
The microsatellite locus to be checked includes：BAT-25, BAT-26, D2S123, D5S346 and D17S250；
The detection primer includes：Shown in the first primer pair, SEQ ID NO.3-4 shown in SEQ ID NO.1-2 Three-primer shown in two primer pairs, SEQ ID NO.5-6 is to the 4th primer pair and SEQ ID shown in, SEQ ID NO.7-8 The 5th primer pair shown in NO.9-10；
The internal reference microsatellite locus is Penta C.
The invention has the advantages that：
The method of colorectal cancer microsatellite instability detection provided by the invention, this method are based on multiple fluorescence PCR technology And capillary electrophoresis technique, with the first primer pair shown in SEQ ID NO.1-2, the second primer shown in SEQ ID NO.3-4 To the three-primer shown in, SEQ ID NO.5-6 to the 4th primer pair and SEQ ID NO.9- shown in, SEQ ID NO.7-8 The 5th primer pair shown in 10 carries out multiplex PCR and detects 5 microsatellite locus BAT-25, BAT-26, D2S123, D5S346 simultaneously And D17S250, there is higher sensitivity and precision, and internal reference microsatellite locus Penta C detection is introduced, for excluding Exogenous DNA pollution in sample to be checked, the precision of the indefinite detection of microsatellite is further improved, improve the confidence level of result.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below by embodiment it is required use it is attached Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is wild plasmid mixture and wild plasmid-mutant plasmids mixture in the embodiment of the present invention 1 Peak type collection of illustrative plates；
Fig. 2 is the peak type collection of illustrative plates to SA-1 pattern detections in the embodiment of the present invention 2；
Fig. 3 is the peak type collection of illustrative plates to SA-2 pattern detections in the embodiment of the present invention 2；
Fig. 4 is the peak type collection of illustrative plates to SA-3 pattern detections in the embodiment of the present invention 2；
Fig. 5 is the peak type collection of illustrative plates to SA-4 pattern detections in the embodiment of the present invention 2；
Fig. 6 is the peak type collection of illustrative plates to SA-5 pattern detections in the embodiment of the present invention 2.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase Product.
The method detected below to the colorectal cancer microsatellite instability of the embodiment of the present invention is specifically described.
The method of colorectal cancer microsatellite instability detection provided in an embodiment of the present invention, it includes：It is to be checked with primer pair Sample enters performing PCR amplification；
Wherein, the primer is included for the detection primer of microsatellite locus to be checked amplification and for internal reference microsatellite locus The internal control primer of amplification；
The microsatellite locus to be checked includes：BAT-25, BAT-26, D2S123, D5S346 and D17S250；
The detection primer includes：Shown in the first primer pair, SEQ ID NO.3-4 shown in SEQ ID NO.1-2 Three-primer shown in two primer pairs, SEQ ID NO.5-6 is to the 4th primer pair and SEQ ID shown in, SEQ ID NO.7-8 The 5th primer pair shown in NO.9-10；
The internal reference microsatellite locus is Penta C.
Wherein, the first primer pair detection BAT-25 sites shown in SEQ ID NO.1-2, shown in SEQ ID NO.3-4 Second primer pair detection BAT-26 sites, the three-primer shown in SEQ ID NO.5-6 is to detection D2S123 sites, SEQ ID The 4th primer pair detection D5S346 sites shown in NO.7-8, the 5th primer pair detection shown in SEQ ID NO.9-10 D17S250 sites.
Positions of the Penta C in genome is 9p13, and its repeat unit is [GTTTT]3-15.Penta C compare other STR bit point has the polymorphism of height and a low-level microsatellite instability, and the polymorphism of height becomes people's Identification, ensure that everyone has unique characteristic spectrum.Low-level microsatellite instability meets it as internal reference Stability.
The method of colorectal cancer microsatellite instability detection provided by the invention, this method are based on multiple fluorescence PCR technology With Capillary Electrophoresis (capillary electrophoresis, CE) technology, with the first primer shown in SEQ ID NO.1-2 To the second primer pair shown in, SEQ ID NO.3-4, the three-primer shown in SEQ ID NO.5-6 to, SEQ ID NO.7-8 The 4th shown primer pair and the 5th primer pair shown in SEQ ID NO.9-10 carry out multiplex PCR and detect 5 microsatellites simultaneously Site BAT-25, BAT-26, D2S123, D5S346 and D17S250, there is higher sensitivity and precision, and introduce internal reference Microsatellite locus Penta C detection, for excluding the pollution of the exogenous DNA in sample to be checked, it is indefinite further to improve microsatellite The precision of detection, improve the confidence level of result.
Further, in some embodiments of the present invention, the internal control primer is included shown in SEQ ID NO.11-12 The 6th primer pair.
The 6th primer pair shown in SEQ ID NO.11-12 can be directed to internal reference microsatellite locus Penta C and detect, same In PCR reaction systems, non-specific amplification can be avoided, improves specific amplification, while be favorably improved the reliable of testing result Property.
Further, in some embodiments of the present invention, second primer pair and the 5th primer pair marked First fluorescence labeling；
4th primer pair and the 6th thing are to having marked fluorescence labeling；
First primer pair and three-primer are to having marked three fluorescence labelings；
Both any not phases in first fluorescence labeling, second fluorescence labeling and the 3rd fluorescence labeling Together.
Further, in some embodiments of the present invention, first fluorescence labeling is in following fluorescence labeling Any one：FAM, HEX, JOE, TAMRA, ROX and VIC；
Any one of second fluorescence labeling in following fluorescence labeling：FAM, HEX, JOE, TAMRA, ROX and VIC；
Any one of 3rd fluorescence labeling in following fluorescence labeling：FAM, HEX, JOE, TAMRA, ROX and VIC。
Further, in some embodiments of the present invention, in first primer pair, second primer pair, institute State three-primer to, in the 4th primer pair, the 5th primer pair and the 6th primer pair, the upstream of each primer pair 5 ' ends of primer are marked with fluorescence labeling.
Using three kinds of different fluorescent labels 6 to primer pair so that two kinds of PCR primers for having length adjacent are detecting When show different fluorescence, be favorably improved resolution ratio, be easy to read testing result, avoid misreading.
It should be noted that fluorescence labeling can mark the sense primer in primer pair 5 ' hold or anti-sense primer 5 ' End, it is some in particular cases (such as need strengthen fluorescence signal when), can also by the sense primer in primer pair and under Swim the equal mark fluorescent mark in 5 ' ends of primer.
With fluorescence labeling primer amplification product can be used capillary electrophoresis technique detected, improve resolution ratio and The precision of testing result.
Further, in some embodiments of the present invention, in the system of PCR amplifications, first primer pair Concentration is 0.0125-0.05 μM, and the concentration of second primer pair is 0.05-0.2 μM, and the concentration of the three-primer pair is 0.1-0.4 μM, the concentration of the 4th primer pair is 0.1-0.4 μM, and the concentration of the 5th primer pair is 0.1-0.4 μM, institute The concentration for stating the 6th primer pair is 0.025-0.1 μM.
Further, at least one embodiment of the present invention, in the system of PCR amplifications, first primer To concentration be 0.025 μM, the concentration of second primer pair is 0.1 μM, and the concentration of the three-primer pair is 0.2 μM, institute The concentration for stating the 4th primer pair is 0.2 μM, and the concentration of the 5th primer pair is 0.2 μM, and the concentration of the 6th primer pair is 0.05μM。
Further, in some embodiments of the present invention, the condition of PCR amplifications is：94-96 DEG C of pre-degeneration 3- 5min；94-96 DEG C of denaturation 25-45s；58-60 DEG C of annealing 60-120s, 70-72 DEG C of extension 30-60s, 29-32 circulates；58-60 DEG C extension 30-60min.
Further, at least one embodiment of the present invention, the condition of PCR amplifications is：95 DEG C of pre-degeneration 4min； 95 DEG C of denaturation 30s；60 DEG C of annealing 90s, 72 DEG C of extension 30s, 30 circulate；60 DEG C of extension 45min.
Further, in some embodiments of the present invention, following components is also contained in the system of PCR amplifications：Tris- HCl、KCl、(NH4)2SO4、dNTP、MgCl2And glycerine.
Further, in some embodiments of the present invention, in the system of PCR amplifications, Tris-HCl concentration is 18-22mM, KCl concentration are 18-22mM, (NH4)2SO4Concentration be that 3-7mM, dNTP concentration is 0.1-0.5mM, MgCl2's Concentration is 2-6mM, the mass percent of glycerine is 1-2%.
Further, at least one embodiment of the present invention, in the system of PCR amplifications, Tris-HCl's is dense To spend for 20mM, KCl concentration be 20mM, (NH4)2SO4Concentration be that 5mM, dNTP concentration is 0.3mM, MgCl2Concentration be 4mM, the mass percent of glycerine is 1.5%.
Further, in some embodiments of the present invention, methods described also includes：The product of PCR amplifications is carried out Capillary Electrophoresis.
Further, in some embodiments of the invention, measuring samples are tumor sample, wherein, tumor sample is swollen Oncocyte, tumor tissues or tumor tissues paraffin section.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment with the wild plasmid to structure and mutant plasmids to the colorectal cancer microsatellite of the present embodiment not The method of stable detection illustrates, specific as follows.
1 wild plasmid is built
Using the blood sample genomic DNA of people as template, using above-mentioned six sites (BAT-25, BAT-26, D2S123, D5S346 and D17S250) unstressed configuration mark primer (the first primer pair, SEQ ID NO.3- shown in SEQ ID NO.1-2 The three-primer shown in the second primer pair, SEQ ID NO.5-6 shown in 4 is to the 4th primer shown in, SEQ ID NO.7-8 To the 5th primer pair shown in, SEQ ID NO.9-10 and the 6th primer pair shown in SEQ ID NO.11-12) carry out respectively PCR is expanded, and amplified production is using PMD18-T vector Cloning Kit (numberings：6011, TaKaRa) TA clones are carried out, so Picking colony PCR is accredited as correct positive clone molecule expansion culture, plasmid extraction afterwards, and the positive plasmid of extracting delivers to raw work life The sequencing portion sequencing of thing engineering (Shanghai) limited company.The correct plasmid of sequence verification is retained as wild type mixing plasmid Base unit and mutant plasmids structure template.
2 mutant plasmids are built
Five in addition to Penta C microsatellite locus with above-mentioned structure is BAT-25, BAT-26, D2S123, Wild plasmid corresponding to D5S346 and D17S250 is template, the primer of rite-directed mutagenesis is designed, using Site-directed Mutagenesis Kit (production code members：B639281-0020 rite-directed mutagenesis experiment) is carried out, obtains the purpose plasmid of saltant type. The following institute of fragment sequence inserted in the wild type and mutant plasmids and Penta C wild plasmid in five sites to be checked Show：
BAT-25 detection sites wild plasmid (Wild plasmid (BAT-25))：
ACTATGGCTCTAAAATGCTCTGTTCTCAAAAAAAAAAAAAAAAAAAAAAAAAATCAAAAAAACAAAACACAAAACTC TTTAGAGAATCACTCCCACTTACATTCTTGGAGGCGAGGAAAG；
BAT-25 detection sites mutant plasmids (Mutation plasmid (BAT-25))：
ACTATGGCTCTAAAATGCTCTGTTCTCAAAAAAAAAAAAAAAAAAAAATCAAAAAAACAAAACACAAAACTCTTTAG AGAATCACTCCCACTTACATTCTTGGAGGCGAGGAAAG；
BAT-26 detection sites wild plasmid (Wild plasmid (BAT-26))：
CAGAGCCCTTAACCTTTTTCAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTTAAAAATGTTGAATGGTTAAA AAATGTTTTCATTGACATATACTGAAGAAGC；
BAT-26 detection sites mutant plasmids (Mutation plasmid (BAT-26))：
CAGAGCCCTTAACCTTTTTCAGGTAAAAAAAAAAAAAAAAAAAAAAAAGGGTTAAAAATGTTGAATGGTTAAAAAAT GTTTTCATTGACATATACTGAAGAAGC；
D2S123 detection sites wild plasmid (Wild plasmid (D2S123))：
ATGCCTGCCTTTAACACTGCTATTCAACATTGCTGGAAGTTCTGGCCAGAGAAATTAGACACAGTGATACACACACA CACACACACACACACACACACACACACACACACACACATATTTTATAGATAGATAGATGGTATCCAAGTCAGAAAGG GAGAAGTAAAACTATCCCTATTGTAGATGACACAGTCCCA；
D2S123 detection sites mutant plasmids (Mutation plasmid (D2S123))：
ATGCCTGCCTTTAACACTGCTATTCAACATTGCTGGAAGTTCTGGCCAGAGAAATTAGACACAGTGATCACACACAC ACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACATATTTTATAGATAG ATAGATGGTATCCAAGTCAGAAAGGGAGAAGTAAAACTATCCCTATTGTAGATGACACAGTCCCA；
D5S346 detection sites wild plasmid (Wild plasmid (D5S346))：
TCAGGGAATTGAGAGTTACAGGTTACTCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAATTTCCCGATTTATCAC TAGAGTGAGTAACTAACTAACTAACTGCTTTATAAAGCTATCCTGGTATTCATATGCCA；
D5S346 detection sites mutant plasmids (Mutation plasmid (D5S346))：
TCAGGGAATTGAGAGTTACAGGTTACTCGTGTGTGTGTGTGTAAATTTCCCGATTTATCACTAGAGTGAGTAACTAA CTAACTAACTGCTTTATAAAGCTATCCTGGTATTCATATGCCA；
D17S250 detection sites wild plasmid (Wild plasmid (D17S250))：
TCAGCTGGCCATATATATATTTAAACCATTTGAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTT GAAACCATTTGAAAGTTTATGTATGTGTATATATATATATAAACACACACATATTTTTATTGTCTATTTGATTCTTC CTT；
D17S250 detection sites mutant plasmids (mutation plasmid (D17S250))：
TCAGCTGGCCATATATATATTTAAACCATTTGAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGAAACCATTT GAAAGTTTATGTATGTGTATATATATATATAAACACACACATATTTTTATTGTCTATTTGATTCTTCCTT；
Penta C internal references site wild plasmid (Wild plasmid (Penta C))：
CAGGGATATGCACTGGTAATAGAAAAGAGGGACTAAGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG TTTTGTTTTTCTGAAGAAGTCCCTAGAAGCGCTCAGTGTTGGAATGCTCTCTTGTAGCAGTGGCGGCTGCTGCTGGT TCCGGGTCAGATGC；
Wherein, underscore is repetition unit sequence.
The preparation of 3 wild plasmid mixtures and mutant plasmids mixture
The wild plasmid in each site of above-mentioned acquisition is diluted to 10ng/ μ l according to content shown in table 1, according to required Total amount adjust the wild plasmid and Nuclease-free ddH in each site2O dosage, is sufficiently mixed.Then will obtain Wild plasmid mixture dilutes 1000 times as the wild type detection of colorectal cancer microsatellite instability detection (MSI detections) Template.
The wild plasmid mixture of table 1 (100 μ l) matches somebody with somebody tabulation
The wild plasmid in each site of above-mentioned acquisition and mutant plasmids are diluted to accordingly according to method shown in table 2 Concentration, adjust each wild plasmid, mutant plasmids and Nuclease-free ddH according to required total amount2O use Amount, is sufficiently mixed.Then wild plasmid-mutant plasmids mixture will be obtained and dilute 1000 times of mutation as MSI detections The template of type detection.
2 wild plasmids of table-mutant plasmids mixture (100 μ l) match somebody with somebody tabulation
Wherein, the wild plasmid in each site and mutant plasmids base difference size are as shown in table 3.
The wild plasmid of table 3 and mutant plasmids difference base statistical form
4MSI detects the foundation of reaction system and reaction condition
Respectively using above-mentioned acquisition wild plasmid mixture and wild plasmid-mutant plasmids mixture as template, build Reaction system under Liru：
Wherein, 2 × MSI PCR Master Mix include：2×Hot Start PCR Buffer,0.6mM dNTP,8mM MgCl2 and 3%Glycerol；
2 × Hot Start PCR Buffer include：40mM Tris-HCl (pH 8.3 under the conditions of 25 DEG C), 40mM KCl and 10mM (NH4)2SO4。
The primer concentration of each microsatellite locus to be checked is respectively in 5 × MSI primer mix：
0.125μM BAT-25-F/R(ROX)；
0.5μM BAT-26-F(FAM)/R；
1μM D2S123-F(ROX)/R；
1μM D5S346-F(JOE)/R；
1 μM of D17S250-F (FAM)/R and 0.25 μM Penta C-F (JOE)/R.
Each above-mentioned primer sequence information is as shown in table 3 below.
Table 3
MSI detections reaction system is run according to following program：
5MSI detects the CE electrophoresis of reaction product
(1) by GeneScanTM500LIZ Size Standard and Hi-DiTMFormamide is with 1.5:100 ratio It is sufficiently mixed, takes 9 μ l mixtures to be added in 96 orifice plates, adds 1 μ l multiple fluorescence PCR products, mixing is stood several minutes, of short duration It is put into after centrifugation on ABI 3730XL sequenators, prepares detection.
(2) ABI 3730XL sequenator data acquisition softwares, the plate mark of the upper machine testing of editor are opened, importing detects program point Hit operation, you can start to detect.
(3) after detection finishes, data are preserved for subsequent data analysis.
6 microsatellite instabilities detect CE electrophoresis result data analyses, and operation is with reference to as follows：
(1) initial data is imported.Selection " the New Project ", then " project type " are right in File menus Talk about in frame and select " Microsatellite ".Click on and " Add sample to project ", choose file where sample to be analyzed Folder, add to list are clicked on, click on the add on the right side of dialog box, complete the importing of initial data.
(2) selection analysis parameter.Selected respectively in corresponding analysis Method and Size Standard columns LIZ Size Standard used in Microsatellite Default and CE electrophoresis.
(3) data analysis." analysis " button is clicked on to carry out data analysis or click on menu " Analysis- Analyze ", " filename of preservation is inputted in Save Project " dialog boxes, can be automatically analyzed after preservation in appearance.
(4) low quality data reanalyses.Selected after analysis " Analysis-low Quality To Top ", so All low quality samples (SQ is red or yellow sign) are chosen afterwards, select " Analysis-Size Match Editor ", " Editor-Delete All Size Labels " or the dialog box in appearance are selected in the dialog box of appearance " Editor-Override All SQ " click on " OK ", then repeat a data analysis for middle selection.
(5) adjustment of sample order.Sampfe order can be adjusted for convenience of data analysis, selects " Tools-Table Setting ", the information " Samples " and genotype " Genotypes " of sample are adjusted in the dialog box of appearance, point Hit " OK ".
(6) display of fluorescence labeling color.The Maker to be analyzed is chosen, selects " Analysis-Display Polts " Or shortcut " Ctrl+L "." the Samples Plot " pages are adjusted to the Maker of analysis parameter.Toolbar " Plot Setting " are the type of analysis；" Panes " is the number for the Maker that the page is shown, is adjusted as requested；Root Corresponding color is selected to show according to the fluorescence of Maker marks, the dye colour of general FAM marks is blue, JOE and HEX marks Dye colour be green, TRAMA mark dye colour be yellow (in order to eye-catching, showing black in software), ROX mark Dye colour for it is red, orange generally internal reference dye colour.Corresponding color is selected according to Maker marks or filtered Other colors.
(7) adjustment of the abscissa and ordinate of peak type figure." View " function of menu bar is selected according to demand, it is right The abscissa of Maker peak figures and the sign of ordinate are adjusted.
(8) adjustment of field range.The abscissa and ordinate scope shown accordingly according to clip size selection.To Abscissa fragment length is adjusted, certain scope is chosen when cursor shows different；To adjust ordinate, then light is needed Click right on the vertical scale is marked, modify " Full view or Zoom to ".
(9) lookup of peak type figure information.If it is to be understood that the specifying information at a certain peak needs a Table, need to select In the peak, selection " View-Table filter-Show Selected Rows ", then " Export Table ", the lattice of output Formula is that " TXT " then needs to be converted into Excel forms.
(10) specific data analysis process refers to GeneMapper software specifications.
It should be noted that in other examples, it is possible to use other instruments are analyzed CE electrophoresis results, Related operating procedure is carried out according to specification.
7 result interpretations
As can be seen from Figure 1 (in figure：Site corresponding to peak type collection of illustrative plates from left to right is followed successively by 1：BAT-26,2：BAT-25,3： D5S346,4：D17S250,5：D2S123,6：Penta C；A- represents the peak type collection of illustrative plates of wild plasmid mixture, and B- represents open country The peak type collection of illustrative plates of raw type plasmid-mutant plasmids mixture), the MSI using wild plasmid-mutant plasmids mixture as template The peak type trace analysis analysis of detection understands that BAT-26, BAT-25, D5S346, D17S250 and five sites to be checked of D2S123 are all There is bimodal (Figure 1B), illustrate that the present invention can accurately detect to whether there is same microsatellite locus not in sample Same repeat unit, its test limit can as little as 0.05pg/ μ l, thus illustrate the present embodiment colorectal cancer microsatellite instability detection Method there is higher sensitivity.
Embodiment 2
The present embodiment is cut into slices as measuring samples using the paraffin-embedded tissue from 5 colorectal cancer patients, to this implementation The method for the colorectal cancer microsatellite instability detection that example provides illustrates, specific as follows.
The paraffin-embedded tissue slice number of five colorectal cancer patients is respectively：SA-1, SA-2, SA-3, SA-4, SA- 5, while control is used as using the MSI analysis system v1.2 of Promega companies, and enter in strict accordance with its specification Row operation.
1DNA is extracted
Using paraffin-embedded tissue DNA rapid extractions kit (TIANGEN Biotech (Beijing) Co., Ltd.) respectively to five The colorectal cancer paraffin-embedded tissue section of position patient and cancer beside organism's specimens paraffin embedding slices carry out genome DNA extraction, strictly Operated to specifications, the genomic DNA of extracting using micro-spectrophotometer quantitatively after, draw a part and be diluted to 5ng/μl。
2MSI detects the foundation of reaction system and reaction condition
It is essentially identical with the step 4 of embodiment 1, the difference is that with one group of genome of every colorectal cancer patients of acquisition DNA is template.
The multiple fluorescence PCR products CE electrophoresis of 3 microsatellite instabilities detection
With the step 5 of embodiment 1.
4 microsatellite instabilities detect CE electrophoresis result data analyses
With the step 6 of embodiment 1.
5 result interpretations
As shown in figures 2-6 (in figure：Abscissa represents base size, and ordinate represents fluorescence signal intensity.From left to right Site corresponding to peak type collection of illustrative plates is followed successively by BAT-26, BAT-25, D5S346, D17S250, D2S123, Penta C；Promega pairs NR-21, BAT-26, BAT-25, NR-24, MONO-27 are followed successively by according to site corresponding to the peak type collection of illustrative plates of kit from left to right, Penta C), the peak figure of each sample has appearance in Penta C, illustrates that measuring samples are not contaminated, testing result is credible；
Numbering is that five detection site peak type collection of illustrative plates of the sample of tetra- patients of SA-1, SA-2, SA-3 and SA-4 are not all sent out Changing, it is MSS types to judge this four patients.
Five site spectrograms to be checked of the sample for the patient that numbering is SA-5 have tri- positions of BAT-26, BAT-25 and D2S123 Point appearance is bimodal, judges the patient for MSI-H types.
The peak type collection of illustrative plates that Promega kits detect the MSI states of five colorectal cancer patients shows that numbering is SA-1, Five detection site peak type collection of illustrative plates of tetra- patients of SA-2, SA-3 and SA-4 all do not change, and judge that this four patients are MSS Type, numbering are that tetra- sites of NR-21, BAT-26, BAT-25 and MONO-27 of SA-5 patient change, and are determined as MSI-H Type.Five patients are consistent with the testing result of Promega contrast agents boxes using the testing result of the present invention.It is described above, this The method precision for the colorectal cancer microsatellite instability detection that inventive embodiments provide is high, and testing result is reliable.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
SEQUENCE LISTING
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Claims (10)

  1. A kind of 1. method of colorectal cancer microsatellite instability detection, it is characterised in that it includes：Entered with primer pair measuring samples Performing PCR expands；
    Wherein, the primer includes expanding for the detection primer of microsatellite locus to be checked amplification and for internal reference microsatellite locus Internal control primer；
    The microsatellite locus to be checked includes：BAT-25, BAT-26, D2S123, D5S346 and D17S250；
    The detection primer includes：Second shown in the first primer pair, SEQ ID NO.3-4 shown in SEQ ID NO.1-2 is drawn Thing is to the three-primer shown in, SEQ ID NO.5-6 to the 4th primer pair and SEQ ID shown in, SEQ ID NO.7-8 The 5th primer pair shown in NO.9-10；
    The internal reference microsatellite locus is Penta C.
  2. 2. the method for colorectal cancer microsatellite instability detection according to claim 1, it is characterised in that the interior reference Thing includes the 6th primer pair shown in SEQ ID NO.11-12.
  3. 3. the method for colorectal cancer microsatellite instability detection according to claim 2, it is characterised in that described second draws Thing pair and the 5th primer pair have marked fluorescence labeling；
    4th primer pair and the 6th thing are to having marked fluorescence labeling；
    First primer pair and three-primer are to having marked three fluorescence labelings；
    In first fluorescence labeling, second fluorescence labeling and the 3rd fluorescence labeling it is any both differ.
  4. 4. the method for colorectal cancer microsatellite instability detection according to claim 3, it is characterised in that described first is glimmering Any one of signal in following fluorescence labeling：FAM, HEX, JOE, TAMRA, ROX and VIC；
    Any one of second fluorescence labeling in following fluorescence labeling：FAM, HEX, JOE, TAMRA, ROX and VIC；
    Any one of 3rd fluorescence labeling in following fluorescence labeling：FAM, HEX, JOE, TAMRA, ROX and VIC.
  5. 5. the method for colorectal cancer microsatellite instability detection according to claim 4, it is characterised in that described first Primer pair, second primer pair, the three-primer to, the 4th primer pair, the 5th primer pair and the described 6th In primer pair, 5 ' ends of the sense primer of each primer pair are marked with fluorescence labeling.
  6. 6. the method for the colorectal cancer microsatellite instability detection according to claim any one of 3-5, it is characterised in that In the system of PCR amplifications, the concentration of first primer pair is 0.0125-0.05 μM, and the concentration of second primer pair is 0.05-0.2 μM, the concentration of the three-primer pair is 0.1-0.4 μM, and the concentration of the 4th primer pair is 0.1-0.4 μM, institute The concentration for stating the 5th primer pair is 0.1-0.4 μM, and the concentration of the 6th primer pair is 0.025-0.1 μM.
  7. 7. the method for colorectal cancer microsatellite instability detection according to claim 6, it is characterised in that PCR amplifications Condition is：94-96 DEG C of pre-degeneration 3-5min；94-96 DEG C of denaturation 25-45s；58-60 DEG C of annealing 60-120s, 70-72 DEG C of extension 30-60s, 29-32 circulations；58-60 DEG C of extension 30-60min.
  8. 8. the method for the colorectal cancer microsatellite instability detection according to claim any one of 3-5, it is characterised in that Also contain following components in the system of PCR amplifications：Tris-HCl、KCl、(NH4)2SO4、dNTP、MgCl2And glycerine.
  9. 9. the method for colorectal cancer microsatellite instability detection according to claim 8, it is characterised in that expanded in PCR System in, Tris-HCl concentration be 18-22mM, KCl concentration be 18-22mM, (NH4)2SO4Concentration be 3-7mM, dNTP Concentration be 0.1-0.5mM, MgCl2Concentration be 2-6mM, the mass percent of glycerine be 1-2%.
  10. 10. the method for the colorectal cancer microsatellite instability detection according to claim any one of 1-5, it is characterised in that Methods described also includes：The product of PCR amplifications is subjected to Capillary Electrophoresis.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305958A (en) * 2018-11-23 2019-10-08 郑州海普生物医药科技有限公司 A kind of MSI detection method
CN110699455A (en) * 2019-10-29 2020-01-17 苏州绘真医学检验有限公司 Human circulating tumor cell MSI detection primer group, kit and detection method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030113723A1 (en) * 2000-10-04 2003-06-19 Bharati Bapat Method for evaluating microsatellite instability in a tumor sample
CN107217103A (en) * 2017-07-14 2017-09-29 常州桐树生物科技有限公司 Detect the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030113723A1 (en) * 2000-10-04 2003-06-19 Bharati Bapat Method for evaluating microsatellite instability in a tumor sample
CN107217103A (en) * 2017-07-14 2017-09-29 常州桐树生物科技有限公司 Detect the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305958A (en) * 2018-11-23 2019-10-08 郑州海普生物医药科技有限公司 A kind of MSI detection method
CN110699455A (en) * 2019-10-29 2020-01-17 苏州绘真医学检验有限公司 Human circulating tumor cell MSI detection primer group, kit and detection method

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