CN106757379B - Lung cancer polygenic variation library constructing method - Google Patents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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Abstract
The present invention relates to a kind of lung cancer polygenic variation library constructing methods, include the following steps：A. according to the variable region of lung cancer related gene, design can detect the primer pair of these variable regions；B. the judgement parameter R of each primer pair is calculated, will determine that the value of parameter R is less than or equal to 1 primer pair, is classified as first group of primer combination liquid, will determine that the value of parameter R is more than 1 primer pair, is classified as second group of primer combination liquid；C. by sample to be tested DNA extracting and purifyings；D. first group of primer combination liquid is respectively adopted and second group of primer combination liquid carries out initial PCR amplification to sample after purification；E. connector is carried out to the target fragment to connect to obtain belt lacing segment；F. it uses first group of primer to combine the mixed liquor progress Library PCR amplification of liquid and second group of primer combination liquid, obtains sequencing library.Library sequencing throughput constructed by the lung cancer polygenic variation library constructing method of the present invention is high, and sensitivity is strong, high specificity, can be used in detecting the mutation of dissociative DNA low frequency.
Description
Technical field
The present invention relates to a kind of lung cancer polygenic variation library constructing methods, belong to biotechnology.
Background technology
The morbidity and mortality of tumour are higher than other diseases at present, and exploitation carries out tumour base with non-invasive diagnosing strategy
Because diagnosis is researched and developed, for Preventive and drug resistance after tumor targets and chemotherapy medication, and traditional medical diagnosis on disease is passed through by clinic
Test, pathological monitoring, cell detection are as foundation, there is the limitation of sensitivity, can not also accomplish early detection early treatment or ahead of time
Prediction prevents ahead of time.
High throughput technique of gene detection of new generation, now mainly with the Hiseq of Illumina companies, Miseq series and
The Ion Torrent of Life companiesTMSerial sequenator is representative, the high throughput for having other technologies incomparable, low cost etc.
Advantage.Have been widely used for different kind organism research nuclear medicine detection.To there is the species sequence of reference, carries out full-length genome and resurvey
Sequence detects mutational site in full-length genome level, finds the molecular basis of individual difference.New-generation sequencing technology passes through full base
Because group sequencing constructs the Genome Atlas of wide variety of conventional species, also promote sequencing technologies tells development.Meanwhile it is high-throughput
Sequencing technologies also have important role, the tumor susceptibility gene of certain diseases usually only to account for base in terms of gene diagnosis and gene therapy
Because of a group small part, the research of these tumor susceptibility genes is not needed to genome sequencing, and is only needed to specific
Several genes are studied, the appearance of target sequence capture sequencing so that be possibly realized to the research of genome top region.This
Item technology synthesizes the oligonucleotide sequence that can be largely combined with specific genome area complementation first, and mesh is enriched with by PCR amplification
Section is marked, then these sections are sequenced with high throughput sequencing technologies.
SNPs (single nucleotide polymorphism) refers to the same chromosome in Different Individual or the nucleotide sequence in same site
In, always, and only there are one the different phenomenons of base for most nucleotide sequences.SNP is that change common in human DNA is special-shaped
Formula, millions of there are in the genome of the entire mankind, part SNP can result in the change of protein amino acid encoding or gene table
Up to controlling changing.Copy number variation (CNV, Copy number variations) refers to be widely present in human genome,
The variation of missing, insertion, repetition and complicated multidigit point out of from 1Kbp to millions of bp.SNPs and CNVs is human phenotype
Two important potential sources of variation, are compared with genome reference sequences, are had nothing in common with each other degree with gene expression shape
It is significantly correlated.More at present to the association study of SNPs, many CNVs are located at the complex region of gene structure variation, some CNVs
There are linkage disequilibriums with neighbouring SNPs, can be by examining SNPs genotype to speculate neighbouring CNVs.Preferably each sample
The analysis that this DNA can be integrated with one detects SNPs and CNVs simultaneously, and amplicon sequencing can be according to researcher's purpose, for target
Region carries out the sequencing of high coverage, can also detect that low frequency is mutated.It is sequenced using amplicon, researcher can be to genome
In interested key area carry out primary study.
Lung cancer is most common malignant tumour in world wide, and the incidence of lung cancer becomes in majority state in apparent rise
Gesture, and constantly increase year by year, it is one kind of most common cancer all over the world, incidence and the death rate account for the head of various malignant tumours
Position.Therefore it studies and finds to occur with lung cancer, develop early detection, early diagnosis and treatment side of the relevant factor for lung cancer
The judgement of case drafted with prognosis has great importance.
Currently, resting on individual gene or the detection water in several sites for the diagnostic method majority of lung cancer genetic test
Flat, detection method is mostly fluorescent PCR, chip method etc., but there are different defects, such as complicated for operation, primer or spies for this method
It is more that needle design is complicated, result is not easy interpretation, poor repeatability, false positive and false negative, detection sensitivity is low, Polymorphism number compared with
Less, flux is low etc., cannot meet the actual demand of clinical multisample multidigit point screening, diagnosis and detection, to make tumor patient
Best treatment period is missed.And the consistency of multiplexed PCR amplification efficiency is always hang-up.Amplicon is more, uneven
One property is higher.This uses the sequencing of the range more than 500Kb, it is difficult to be carried out with the method that amplicon is sequenced.
Frontier fast-developing in recent years using deep sequencing detection CNV, the resolution ratio and accuracy rate of CNV detections with
It the increase of sequencing depth and improves.Compared with chip technology, under conditions of sequencing depth enough, it can obtain more accurate
True CNV endpoint locations.Also, by deep sequencing technology, the non-detectable inversion of genetic chip institute and insertion can be detected
Equal genome mutations form can be within the scope of full-length genome with single base since deep sequencing technology is without designing probe
Resolution ratio detects CNV, thus can significantly improve the detection number of CNV.
It can meet the required template secondary library of amplified library to obtain, need polypropylene through postdigestive amplified production
The gel electrophoresis of acyl ammonia or agarose gel purification remove indigested nucleic acid, postdigestive fragment, nuclease etc., it is seen that at present
There are low output or the defects of poor specificity for common secondary library preparation method.
Invention content
The technical problem to be solved in the present invention is to provide a kind of sequencing throughput height, and sensitivity is strong, high specificity, are based on for two generations
Microarray dataset technology can be used in the lung cancer polygenic variation library constructing method for detecting the mutation of dissociative DNA low frequency.
A kind of technical solution that the present invention proposes to solve above-mentioned technical problem is：A kind of lung cancer polygenic variation library structure
Construction method includes the following steps：
A. according to the variable region of lung cancer related gene, design can detect the primer pair of these variable regions；
B. parameter W1, Tm1, W2 and Tm2 of each primer pair are obtained by primer-design software, to which each primer be calculated
To judgement parameter R, calculation formula is as follows：
R=0.1 × [W1+Tm1/ (Tm1+Tm2)]+0.9 × [W2+Tm2/ (Tm1+Tm2)]；
Wherein, W1 is the G/C content of amplified production, and Tm1 is the annealing temperature of amplified production, and W2 is sense primer G/C content
With the average value of downstream primer G/C content, Tm2 is the average value of sense primer annealing temperature and downstream primer annealing temperature,
It will determine that the value of parameter R is less than or equal to 1 primer pair, be classified as first group of primer combination liquid, will determine that parameter R's
Primer pair of the value more than 1 is classified as second group of primer combination liquid；
C. by sample to be tested DNA extracting and purifyings；
D. first group of primer combination liquid is respectively adopted and second group of primer combination liquid carries out initial p CR to sample after purification
Amplification obtains target fragment, then removes primer, and annealing temperature when liquid carries out initial PCR amplification is combined using first group of primer
Less than the annealing temperature combined using second group of primer when liquid carries out initial PCR amplification；
E. it carries out connector to the target fragment to connect to obtain belt lacing segment, then be purified；
F. first group of primer is used to combine the mixed liquor of liquid and second group of primer combination liquid to belt lacing segment after purification
Library PCR amplification is carried out, is then purified, obtains sequencing library.
Above-mentioned lung cancer related gene include AKT1, ALK, BRAF, BRCA1, BRCA2, CDKN2A, C-met, DDR2,
ERBB3, FGFR2, FGFR4, HER2, KRAS, MAP2K1, mTOR, NRAS, NRF2, PIK3CA, PTCH1, RET, SMO, STK11,
TSC1, TSC2, it is one or more in EGFR, TP53, PTEN, FGFR1；Wherein AKT1, ALK, BRAF, BRCA1, BRCA2,
CDKN2A、C-met、DDR2、ERBB3、FGFR2、FGFR4、HER2、KRAS、MAP2K1、mTOR、NRAS、NRF2、PIK3CA、
The variation sequencing type of PTCH1, RET, SMO, STK11, TSC1, TSC2 are mutation sequencings, and the variation of EGFR, TP53, PTEN are surveyed
Sequence type is exon sequencing；The variation sequencing type of C-met, HER2, FGFR1, PIK3CA are amplification sequencings.
The variable region that above-mentioned primer pair is covered includes chr1：162754624-162754667、chr1：
162754761-162754855、chr1：162759815-162759873、chr1：162775671-162775740、chr1：
162778592-162778698、chr14:104770390-104770420、chr14:104772927-104772980、
chr14:104773502-104773537、chr14:104773920-104773953、chr14:104775128-
104775143、chr14:104776719-104776747、chr14:104780114-104780142、chr14:
104792600-92637、chr2:29193886-29193891、chr2:29196820-29196858、chr2:29197575-
29197633、chr2:29214035-29214042、chr2:29223520-29223528、chr2:29225500-
29225518、chr2:29226922-29226934、chr2:29296963-29296968、chr2:29318371-
29318380、chr2:29328373-29328388、chr2:29333767-29333795、chr2:29694861-
29694888、chr2:29920019-29920086、chr7:140734607-140734688、chr7:140753301-
140753370、chr17：43082440-43082543、chr17：43092005-43092100、chr17：43092907-
43092953、chr17：43093990-43094074、chr17：43106475-43106492、chr17：43115753-
43115774、chr13：32326505-32326575、chr13：32332322-32332386、chr13：32332536-
32332638、chr13：32333139-32333219、chr13：32336459-32336562、chr13：32337435-
32337471、chr13：32363206-32363316、chr13：32398684-32398766、chr9：21971186-
21971208、chr9：21974764-21974803、chr9：21968234-21968234、chr12：56085007-
56085128、chr12：56086541-56086555、chr12：56087577-56087640、chr12：56096505-
56096539、chr12：56097153-56097184、chr12：56101967-56102051、chr10：121479871-
121479899、chr10：121483723-121483769、chr10：121503794-121503854、chr10：
121538612-121538640、chr10：121551339-121551363、chr10：121564503-121564565、
chr10：121593750-121593777、chr5：177089639-17789673、chr5：177093269-177093311、
chr5：177096144-177096163、chr5：177097296-177097324、chr12：25227304-25227374、
chr12：25245285-25245312、chr15：66387410-66387421、chr15：66443280-66443310、
chr15：66490556-66490572、chr1：11117046-11117073、chr1：11130582-11130597、chr1：
11109657-11109693、chr1：11258571-11258588、chr1：114716116-11471652、chr2:
177230956-177230973、chr2:177231581-177231624、chr2:177231869-177231911、chr9:
95446922-95446944、chr9:95453505-95453539、chr9:95459693-95459726、chr9:
95469086-95469107、chr9:95508250-95508271、chr10:43100510-43100560、chr10:
43111228-43111238、chr10:43111342-43111412、chr10:43121969-43121991、chr7:
129189158-129189225、chr7:129210436-129210486、chr7:129212139-129212169、chr19:
1207007-1207038、chr19:1219351-1219462、chr19:1223130-1223160、chr9:132896605-
132896654、chr9:132902616-132902673、chr9:132904419-132904449、chr9:132905730-
132905803、chr9:132906751-132906779、chr9:132928836-132928863、chr16:2048689-
2048723、chr16:2050409-2050486、chr16:2061964-2062005、chr16:2070478-2070523、
chr16:2071878-2071911、chr16:2084534-2084653、chr16:2086217-2086296、chr3:
179198915-179198966、chr3:179199803-179199860、chr3:179203618-179203697、chr3:
179204553-179204586、chr3:179210214-179210276、chr17:39707104-39707141、chr17:
39710358-39710389、chr17:39715774-39715862、chr17:39724825-39724865、chr17:
39726571-39726598、chr17:39727871-39727981、chr7:116731688-116731707、chr7:
116740046-116740083、chr7:116771552-116771574、chr7:116782003-116782070、chr7:
116795915-116795945、chr7:116775003-116775102、chr8:38413973-38414170、chr8:
38415881-38415915、chr8:38417348-38417370、chr8:38429801-38429814、chr8:
38457373-38457422、chr7:55173921-55174039、chr7:55174726-55174810、chr7:
55181301-55181337、chr7:55181365-55181457、chr7:55191729-55191869、chr17:
7669616-7669662、chr17:7673736-7673788、chr17:7673736-7673788、chr17:7673736-
7673831、chr17:7674183-7674253、chr17:7675053-7675108、chr17:7676214-7676245、
chr10:87933035-87933086、chr10:87933124-87933183、chr10:One in 87960993-87961048
It is a or multiple.
It according to volume ratio is 1 ﹕ that mixed liquor in above-mentioned steps F, which is first group of primer combination liquid and second group of primer combination liquid,
1 mixes.
5 ' ends of above-mentioned primer pair introduce deoxyinosine nucleotide base.
In above-mentioned steps D when initial PCR amplification, dimethyl sulfoxide (DMSO) is added in the reaction system, dimethyl sulfoxide (DMSO) is added
Volume accounts for the 3%~7% of reaction system volume.
When above-mentioned steps F Chinese library PCR amplifications, glycerine is added in the reaction system, the volume that glycerine is added accounts for reactant
It is the 5%~8% of volume.
The purifying of belt lacing segment is purified using magnetic bead in above-mentioned steps E, the step F Chinese library PCR amplifications
The purifying of product is purified using magnetic bead.
Experiment condition of first group of primer mixed liquor in initial PCR amplification is in above-mentioned steps D：99 DEG C, 2min, 1
Cycle；99 DEG C, 15s, 55 DEG C, 20s, 72 DEG C, 15 cycles of 30s；
Experiment condition of the second group of primer combination liquid in initial PCR amplification is in above-mentioned steps D：99 DEG C, 2min, 1
Cycle；99 DEG C, 15s, 65 DEG C, 20s, 72 DEG C, 30s, 15 cycles；
Experiment of the mixed liquor of first group of primer combination liquid and first group of primer combination liquid in amplified library in above-mentioned steps F
Condition is：98 DEG C, 2min, 1 cycles；99 DEG C, 15s, 62 DEG C, 1min, 5 cycles.
The present invention has the effect of positive：
(1) present invention provides a kind of lung cancer polygenes library constructing method based on two generation microarray dataset technologies, Neng Goushi
The existing low initial amount of concentration of specimens (being less than 10ng), high throughput, short cycle, low cost build library.Library construction using the present invention
The obtained library of method is sequenced, can be with the copy number in a mutational sites and 4 genes up to a hundred of 27 genes of one-time detection
Variation, the 5000 yuan/sample of cost savings relative to generation sequencing mutation.The library can detect the bases such as SNPs and CNVs simultaneously
Because of variation, by the way that sequencing result analysis prognosis evaluation can be carried out to patients with lung cancer.
(2) Primer composition used in the present invention is set for the particular sequence in lung cancer related gene mutational site
Meter introduces deoxyinosine nucleotide base so that interfering with each other property is small between each primer by 5 ' ends.By being in charge of amplification
The selected variable region of capture, is hardly interfered between each other.It is expanded according to judging that Primer composition is in charge of by parameter R,
Other conditions under the same conditions, the success rate of library construction is increased to 93% by original 70%.The present invention can be used for
Minim DNA library construction, reduces the requirement to detecting sample, and the DNA concentration of sample can be built down to 1ng~10ng with other
Library is compared, and library initial amount reduces 40ng.
(3) gene mutation, gene copy number variation and the goldstandard experimental result that the present invention calculates compare, the sequencing of two generations
Susceptibility and specificity are 98.6% and 99.2% respectively；
(4) present invention requires the DNA concentration of sample very low, it is possible to be suitable for free Circulating tumor DNA and (be directed to lung
The peripheral blood DNA of carninomatosis people, ctDNA), tumour cell purity deficiency, the sampling for saving tissue penetration (biopsy) be not inconvenient, true
Determine risk and measures the sensitivity of gene mutation, coverage problem with conventional method difficulty；With facilitate sampling, can early detection with recurrence
Assessment, easily periodically tracking, highly sensitive, the high precisely advantage of resolution ratio and specificity.
(5) present invention optimizes the reagent used during primary amplification and amplified library, and foundation is most suitable for building library
Reaction condition, compared with existing experimental procedure, the present invention introduces certain a small amount of in library primary amplification and amplified library
Organic reagent uses glycerine when primary amplification using dimethyl sulfoxide (DMSO), amplified library, organic by being added in reaction step
The success rate of reagent, library construction is increased to 97% by original 93%.
Specific implementation mode
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used
In invention is further explained, it should not be understood as limiting the scope of the invention, person skilled in art can
To make some nonessential modifications and adaptations to the present invention according to aforementioned present invention content.In following embodiments, if not specially
Show that reagent used is that analysis is pure, and agents useful for same can be obtained from commercial channel.The experiment of actual conditions is not specified in text
Method, usually according to normal condition as the Science Press that J. Pehanorm Brookers are write publishes for 2002《Molecular cloning is real
Test guide》Condition described in one book, or according to the condition proposed by manufacturer.Unless otherwise defined, the institute used in text
There are professional and scientific terms to have the same meanings as commonly understood by one of ordinary skill in the art.In addition, it is any similar to described content or
Impartial method and material all can be applied in the present invention.
Main agents：
All reagents are bought from regular producer：Multisource Genomic DNA Miniprep Kit(Axygen)、
First group of primer combination liquid of lung cancer polygenes detection primer and second group of primer combine liquid (the hundred limited public affairs of power lattice biotechnology of Shanghai
Department), 5*Ion AmpliSeqTM HiFi Mix(Life technologies)、FuPa ReagentTM(Life
Technologies), AmPure XP Reagent magnetic beads (Life technologies), Ion PGM Beads (Life
technologies)、PGM Template OT2Solutions(Life technologies)、Platinum PCR Super
Mix High Fidelity(Life technologies)、Library Amplification Primer Mix(Life
Technologies) etc..In reagent and consumptive material and bis- generations of Ion Torrent that upper machine template prepares and upper machine is used in the process, are sequenced
Platform matches, including the parts such as PGM Template OT2kit and Ion 314chip.Fresh match is needed before this method experiment
The reagent of system：(100% ethyl alcohol presses 3 to 75% ethyl alcohol with nuclease-free water:1 ratio is prepared).
Key instrument：
Ion Torrent PGM sequenators,3.0 fluorescent quantitation instruments, supercentrifuge, water-bath, whirlpool concussion
Instrument, refrigerator, autoclave, magnetic frame, liquid-transfering gun, PCR instrument, concussion instrument etc..
The lung cancer polygenic variation library constructing method of the present embodiment, includes the following steps：
A. according to the variable region of lung cancer related gene, design can detect the primer pair of these variable regions；
B. according to the G/C content of primer and product, Tm values, primer pair is divided into two groups, i.e. first group of primer combination liquid and the
Two groups of primers combine liquid；
C. by sample to be tested DNA extracting and purifyings；
D. first group of primer combination liquid and second group of primer combination liquid is used to carry out initial p CR respectively to sample after purification
Amplification, obtains target fragment, then removes primer；
E. it carries out connector to the target fragment to connect to obtain belt lacing segment, then be purified using magnetic bead；
F. first group of primer is used to combine the mixed liquor of liquid and second group of primer combination liquid to belt lacing segment after purification
Library PCR amplification is carried out, is then purified using magnetic bead, obtains sequencing library.
In whole process, gel extraction or the method pair of magnetic bead Piece Selection can also be passed through as requested in any step
DNA fragmentation molecular size is selected, the final library molecule for obtaining required size DNA fragmentation, and is drawn in connector connection
Enter characteristic sequence label, the differentiation for different libraries in being sequenced.With negative sample, quality-control product carries out extensive parallel together
Sequencing, the result that instrument obtains obtain the SNP site information contained in sample through data processing and bioinformatic analysis.Institute
It is normal human gene group DNA to state negative sample, and the quality-control product is the control particles for being added 1%, is a kind of quotient
Product reagent is included in the kit PGM Sequencing OT2kit of upper machine sequencing, as in the sequencing experiment of upper machine
Ginseng carries out assessment and Quality Control, if not comprising 1% control particles as a result, saying in sequencing result to sequencing situation
Bright computer experiment failure, it is as a result unreliable.
One, design of primers.
According to the variable region of lung cancer related gene, design can detect the primer pair of these variable regions.
In the present invention, lung cancer related gene includes but not limited to following gene：
1 lung cancer polygenes information table of table
| Serial number | Gene Name | DNA Locus | mRNA Locus | Type is sequenced |
| 1 | AKT1 | NG_012188.1 | NM_001014431 | Mutation sequencing |
| 2 | ALK | NG_009445.1 | NM_004304 | Mutation sequencing |
| 3 | BRAF | NG_007873.3 | NM_004333 | Mutation sequencing |
| 4 | BRCA1 | NG_005905.2 | NM_007294 | Mutation sequencing |
| 5 | BRCA2 | NG_012772.3 | NM_000059 | Mutation sequencing |
| 6 | CDKN2A | NG_007485.1 | NM_000077 | Mutation sequencing |
| 7 | DDR2 | NG_016290.1 | NM_001014796 | Mutation sequencing |
| 8 | ERBB3 | NG_011529.1 | NM_001005915 | Mutation sequencing |
| 9 | FGFR2 | NG_012449.2 | NM_000141 | Mutation sequencing |
| 10 | FGFR4 | NG_012067.1 | NM_001291980 | Mutation sequencing |
| 11 | KRAS | NG_007524.1 | NM_004985.4 | Mutation sequencing |
| 12 | MAP2K1 | NG_008305.1 | NM_002755 | Mutation sequencing |
| 13 | mTOR | NG_033239.1 | NM_004958 | Mutation sequencing |
| 14 | NRAS | NG_007572.1 | NM_002524 | Mutation sequencing |
| 15 | NRF2 | NC_000002.1 | NM_001145412 | Mutation sequencing |
| 16 | PTCH1 | NG_007664.1 | NM_000264 | Mutation sequencing |
| 17 | RET | NG_007489.1 | NM_020630 | Mutation sequencing |
| 18 | SMO | NG_023340.1 | NM_005631 | Mutation sequencing |
| 19 | STK11 | NG_007460.2 | NM_000455 | Mutation sequencing |
| 20 | TSC1 | NG_012386.1 | NM_000368 | Mutation sequencing |
| 21 | TSC2 | NG_005895.1 | NM_000548 | Mutation sequencing |
| 22 | PIK3CA | NG_012113.2 | NM_006218 | Mutation, amplification sequencing |
| 23 | HER2 | NG_007503.1 | NM_001005862 | Mutation, amplification sequencing |
| 24 | C-met | NG_008996.1 | NM_000245 | Mutation, amplification sequencing |
| 25 | FGFR1 | NG_007729.1 | NM_001174063 | Amplification sequencing |
| 26 | EGFR | NG_007726.3 | NM_005228 | Exon is sequenced |
| 27 | TP53 | NG_017013.2 | NM_000546 | Exon is sequenced |
| 28 | PTEN | NG_007466.2 | NM_000314 | Exon is sequenced |
The present embodiment is designed for detecting these lung cancer related genes according to the variable region of above-mentioned each lung cancer related gene
Variable region with the presence or absence of variation primer pair.Variable region includes the exon region of each gene, or with exon phase
Upstream and downstream 30bp's even includes subregion, or with other relevant sudden change region of lung cancer.Primer pair can cover institute in table 1
There is the variable region that the needs of gene detect.The region covered is as follows：
chr1：162754624-162754667、chr1：162754761-162754855、chr1：162759815-
162759873、chr1：162775671-162775740、chr1：162778592-162778698、chr14:104770390-
104770420、chr14:104772927-104772980、chr14:104773502-104773537、chr14:
104773920-104773953、chr14:104775128-104775143、chr14:104776719-104776747、
chr14:104780114-104780142、chr14:104792600-92637、chr2:29193886-29193891、chr2:
29196820-29196858、chr2:29197575-29197633、chr2:29214035-29214042、chr2:
29223520-29223528、chr2:29225500-29225518、chr2:29226922-29226934、chr2:
29296963-29296968、chr2:29318371-29318380、chr2:29328373-29328388、chr2:
29333767-29333795、chr2:29694861-29694888、chr2:29920019-29920086、chr7:
140734607-140734688、chr7:140753301-140753370、chr17：43082440-43082543、chr17：
43092005-43092100、chr17：43092907-43092953、chr17：43093990-43094074、chr17：
43106475-43106492、chr17：43115753-43115774、chr13：32326505-32326575、chr13：
32332322-32332386、chr13：32332536-32332638、chr13：32333139-32333219、chr13：
32336459-32336562、chr13：32337435-32337471、chr13：32363206-32363316、chr13：
32398684-32398766、chr9：21971186-21971208、chr9：21974764-21974803、chr9：
21968234-21968234、chr12：56085007-56085128、chr12：56086541-56086555、chr12：
56087577-56087640、chr12：56096505-56096539、chr12：56097153-56097184、chr12：
56101967-56102051、chr10：121479871-121479899、chr10：121483723-121483769、chr10：
121503794-121503854、chr10：121538612-121538640、chr10：121551339-121551363、
chr10：121564503-121564565、chr10：121593750-121593777、chr5：177089639-17789673、
chr5：177093269-177093311、chr5：177096144-177096163、chr5：177097296-177097324、
chr12：25227304-25227374、chr12：25245285-25245312、chr15：66387410-66387421、
chr15：66443280-66443310、chr15：66490556-66490572、chr1：11117046-11117073、chr1：
11130582-11130597、chr1：11109657-11109693、chr1：11258571-11258588、chr1：
114716116-11471652、chr2:177230956-177230973、chr2:177231581-177231624、chr2:
177231869-177231911、chr9:95446922-95446944、chr9:95453505-95453539、chr9:
95459693-95459726、chr9:95469086-95469107、chr9:95508250-95508271、chr10:
43100510-43100560、chr10:43111228-43111238、chr10:43111342-43111412、chr10:
43121969-43121991、chr7:129189158-129189225、chr7:129210436-129210486、chr7:
129212139-129212169、chr19:1207007-1207038、chr19:1219351-1219462、chr19:
1223130-1223160、chr9:132896605-132896654、chr9:132902616-132902673、chr9:
132904419-132904449、chr9:132905730-132905803、chr9:132906751-132906779、chr9:
132928836-132928863、chr16:2048689-2048723、chr16:2050409-2050486、chr16:
2061964-2062005、chr16:2070478-2070523、chr16:2071878-2071911、chr16:2084534-
2084653、chr16:2086217-2086296、chr3:179198915-179198966、chr3:179199803-
179199860、chr3:179203618-179203697、chr3:179204553-179204586、chr3:179210214-
179210276、chr17:39707104-39707141、chr17:39710358-39710389、chr17:39715774-
39715862、chr17:39724825-39724865、chr17:39726571-39726598、chr17:39727871-
39727981、chr7:116731688-116731707、chr7:116740046-116740083、chr7:116771552-
116771574、chr7:116782003-116782070、chr7:116795915-116795945、chr7:116775003-
116775102、chr8:38413973-38414170、chr8:38415881-38415915、chr8:38417348-
38417370、chr8:38429801-38429814、chr8:38457373-38457422、chr7:55173921-
55174039、chr7:55174726-55174810、chr7:55181301-55181337、chr7:55181365-
55181457、chr7:55191729-55191869、chr17:7669616-7669662、chr17:7673736-7673788、
chr17:7673736-7673788、chr17:7673736-7673831、chr17:7674183-7674253、chr17:
7675053-7675108、chr17:7676214-7676245、chr10:87933035-87933086、chr10:87933124-
87933183、chr10:87960993-87961048。
Designing and preparing for primer pair in the present embodiment uses existing conventional method.Primer pair packet in the present embodiment
Specific forward primer and specific downstream primer are included, sense primer and downstream primer are being joined according to the variable region covered
Examine genome starting and final position design.Primer is diluted by mother liquor, and mother liquor is limited by hundred power lattice biotechnologys
The dry powder of company's synthesis dilutes, and the ultimate density of primer is 100nM.Primer purity should reach PAGE or HPLC grades, no
Containing miscellaneous band.Nucleotide sequence used is all from the gene order of ncbi database announcement when design, and the product after primer amplification is long
Degree is concentrated mainly between 100bp-500bp.
The nucleotide sequence of primer can be arbitrary known array, and sequence itself does not form hair fastener secondary structure, and G, C
Content accounts for the 30%~70% of base quantity summation, and primer sequence selects the nucleotide sequence of length 18bp~25bp.Using special
The multi-primers design software packet (Primer of Visual OMP, the Multi PLX, ABI of such as DNA soft wares of door
Express etc.) carry out design primer, in order to reduce the formation of the illusion such as primer dimer in amplification process, primer pair
It is shown in amplification procedure by prediction minimum with the parameters such as the interacting of other primers, Tm of each primer pair in primer
Approach as possible, not have serious secondary structure such as hair clip, dimers etc..By using different software, overall merit,
Primer is designed as to target-specific or amplicon specificity as far as possible, and primer is usually grouped into subset so that primer
Between interaction, primer dimer formed and super amplicon reduce to minimum level.
The extension of PCR is to hold to start since primer 3 ', and decides the specificity of PCR product, and 5 ' ends then limit PCR and produce
The length of object.5 ' it is terminal modified after do not influence normal PCR reaction not only, and the primer after modifying can more stably with template
In conjunction with having prodigious convenience for the analysis and further operating of PCR product, base group modification carried out on primer, primer is taken
The modification group of band keeps apart other primers so that in same reaction system, completes to expand while more primers.Primer 5 ' is held
Modification include phosphorylation modification, it is amido modified and introduce deoxyinosine nucleotide base, it is therefore preferable to 5 ' end introducing deoxidations
Inosinic acid base.
The primer pair designed in the present embodiment is the optimal combination primer pair verified by experiment screening repeatedly, can
Realize it is efficient, quick, micro build library, maintain with the library of existing banking process be sequenced flow compatibility flexibly and easily can
For Ion Proton microarray datasets, it is suitable for building the library of Ion Proton, structure library can directly upper machine sequencing.
Primer pair in the present embodiment can be applied directly to different second generation microarray datasets, including but not limited to by
Illumina, Life Technologies (ABI), Roche, Helicos Biotechnologies, Pacific
The instrument of these companies of Biosciences manufacture can equally be well applied to the sequenator of other manufacturer's productions.
Two, primer pair is grouped.
Since the template of amplification is fixed sequence template, when design primer, unavoidably has G/C content and Tm values differ
The primer pair of cause, in order to smoothly build sequencing library, the larger primer pair of these difference is during experiment to experiment condition
Requirement with sample can be stringenter, and experimental result is not necessarily set up the project.
Temperature parameter when annealing temperature is primer and template combination, i.e., when 50% primer and complementary series is shown as
Temperature when double chain DNA molecule, it is the more important factor for influencing PCR specificity, in order to determine better suited annealing temperature, root
According to the judgement parameter R that the relevant parameters such as the G/C content of primer and product, annealing temperature calculate, will accurately be set to more convenient
Primer is divided into two groups after meter synthesis, dilution.Judge that the calculation formula of parameter R is as follows：
R=0.1 × [W1+Tm1/ (Tm1+Tm2)]+0.9 × [W2+Tm2/ (Tm1+Tm2)].
Wherein, W1 is the G/C content of amplified production, and Tm1 is the annealing temperature of amplified production, and W2 is sense primer G/C content
With the average value of downstream primer G/C content, Tm2 is the average value of sense primer annealing temperature and downstream primer annealing temperature.W1、
When W2, Tm1, Tm2 are design primer nucleotide sequences, by multi-primers design software packet, the parameter automatically generated.
It is substituted by the parameter that software package generates, calculation formula obtains the judgement parameter R of all primer pairs, and (R generally retains 5
Position effective digital), wherein judging that the value of parameter R is less than or equal to 1 primer pair, it is classified as first group of primer mixed liquor, judges to join
The value of number R is more than 1 primer pair, is classified as second group of primer mixed liquor.
By taking gene DDR2 as an example, the DNA sequence dna (NG_016290) of the gene is found in ncbi database, finds DNA sequences
Overlay area is detected in row：chr1：162754624-162754667、chr1：162754761-162 754855、chr1：
162759815-162759873、chr1：162775671-162775736、chr1：162778592-162778698；According to soft
The primer pair of part Primer Express design covering destination region, primer pair be designed as far as possible target-specific and and other
Primer does not interfere with each other, and the primer nucleotide sequences after design are as shown in table 2.
2 DDR2 primer sequence tables of table
Primer in table 2 is synthesized by hundred Li Ge Bioisystech Co., Ltd, and 5 ' end introducing deoxidations time of synthetic primer are yellow fast
Purine nucleotide base.
According to the value for judging parameter R, the primer pair of serial number 1,2,4 is classified as first group of primer combination liquid；3 He of serial number
5 primer pair is classified as second group of primer combination liquid.The design of primers of other lung cancer related genes and grouping are with reference to DDR2 genes, most
All primer pairs grouping of design detection lung cancer related gene variable region at last, introduces deoxyinosine nucleotide base
After primer pair is respectively synthesized dilution, first group of primer pair combination liquid and second group of primer combination are configured to according to the result of grouping
Liquid.First group of primer pair combination liquid and second group of primer combination liquid carry out primary amplification reaction, Neng Gouzeng respectively in primary amplification
Add it is each reaction amplified production overall product rate, then primer remove step again by amplified production mixed in equal amounts together into
Row experiment.
Three, sample to be tested DNA extracting and purifyings.
The tissue samples and 5 normal structure samples of 5 patients with lung cancer, the correlation of this 10 samples are detected in the present embodiment
Site has already passed through ARMS verifications.
Detection sample in the present invention is any form of tumor sample that can extract nucleic acid, preferably tissue samples,
Including but not limited to：Whole blood, serum, blood plasma and tissue sample；Tissue sample includes but not limited to：It is paraffin-embedded tissue, fresh
Tissue and frozen section.
3.1 sample DNAs extract
Patients with lung cancer group is extracted using kit (Axygen Multisource Genomic DNA Miniprep Kit)
The DNA of sample is knitted, specific operation is referring to reagent kit product specification.Thermo-Fisher nucleic acid-proteins are used after extracting
Quantitative instrument (NanoDrop 2000) measures concentration of specimens.
3.2 DNA are purified
1) 5ng DNA are taken, Nuclease-free Water to 40 μ L are added；
2) 1.5 times of volume (60 μ L) Ampure beads are added, are placed at room temperature for 5min, then be positioned on magnetic frame about
5min, until clarification；
3) supernatant is abandoned, with 75% alcohol (200 μ L, now with the current) washing 2 times, is dried (should not overdrying)；
4) with 25 μ L Nuclease-free Water dissolvings, Qubit detectable concentrations.
3.3 tissue samples DNA concentrations measure
UsingDNA concentration after 3.0 pairs of extractings measures：
1) 1ul Qubit dsDNA HS Reagent and 199ul Qubit dsDNA HS are added to sample tube
Buffer, whirlpool mixing 4s；
2) 1ul working solutions are abandoned to the sample cell interior suction for having added working solution, is added the DNA sample of 1ul, whirlpool mixing 4s,
Often the final volume of pipe is 200ul；
3) all pipes are protected from light incubation 2 minutes in room temperature；
4) exist" DNA " key is clicked in 3.0 main screens, then dsDNA High Sensitivity is selected to analyze mould
Formula；
5) sample tube is put intoIt in 3.0, closes the lid, clicks read；
6) Calculate Initial initial concentrations are selected, Volume of Sample Used are then selected:1ul, this
When the result that shows be sample initial concentration, unit ng/ul；
7) next sample is read：Take out sample in Qubit3.0 fluophotometers.
Four, target fragment is obtained.
First group of primer combination liquid is respectively adopted and second group of primer combination liquid carries out initial p CR expansions to sample after purification
Increasing obtains target fragment, then removes primer.
4.1 initial PCR amplification
Since initial PCR amplification is multi-PRC reaction, each primer pair competes in amplified reaction with other primer
Limited amount dNTPs, polymerase and other reagents, therefore exist and improved method and composition is needed, to allow selectivity
Multiple target nucleic acid molecules in a group nucleic acid molecules are expanded, while avoiding the shape of non-specific amplification product and primer dimer
At, or the degree of non-specific amplification product and primer dimer minimized, reach more efficient, low concentration detection.This
Organic reagent is added in invention in the reaction system, and organic reagent includes glycerine, olive oil, DMSO (dimethyl sulfoxide (DMSO)), it is preferable that
It is DMSO (dimethyl sulfoxide (DMSO)) in the organic reagent that primary amplification is added.
Liquid is combined using first group of primer of the present embodiment and second group of primer combines liquid, if the concentration of sample is more than
First group of primer, can be combined liquid by 10ng and second group of primer combination liquid mixes and carries out initial PCR amplification to sample.
If the concentration of sample is less than 10ng, it is necessary to first group of primer combination liquid be respectively adopted and second group of primer combination liquid carries out initially
PCR amplification, to ensure the quality in library, below by taking the concentration of sample is less than 10ng as an example.
According to the initial PCR amplification reaction system in table 3, reagent proportioning is carried out.
3 initial PCR amplification reaction system ingredient of table
Reaction system is divided into two pipes, and a tube reaction system combines liquid using first group of primer, and another tube reaction system uses
Second group of primer combines liquid, and other compositions are identical.
A small amount of dimethyl sulfoxide (DMSO) (DMSO) is added in the reaction system, melting temperature can be reduced, primer is contributed to move back
Auxiliary of fighting archaeal dna polymerase extends through secondary structure area, improves primer specificity, effectively facilitate G/C content be up to 52%~
The amplification of 73% template, it is a small amount of to recycle so that not interfere with each other between primer using the primer of low concentration.
It is divided to two pipes to carry out initial PCR amplification respectively and target fragment (target area sequencing target fragment) is obtained by the reaction, uses
The amplified reaction program of first group of primer combination liquid is as shown in table 4, and the amplified reaction program of liquid is combined such as using second group of primer
Shown in table 5.
Table 4 combines the amplified reaction program of liquid using first group of primer
Table 5 combines the amplified reaction program of liquid using second group of primer
PCR reaction conditions：Special primer extends with target sequence annealing, selects higher renaturation temperature to reduce primer and mould
Non-specific binding between plate, it is ensured that the specificity of PCR reactions.The renaturation time slightly extends, so that complete between primer and template
It is complete to combine, but extension of time is unsuitable long, otherwise can lead to the appearance of non-specific amplification band.
4.2 primers are removed
After two pipe amplified reactions obtain target fragment respectively, different amplification target area products is remixed same
In reaction system, library construction step is carried out, the requirement of sample detection is reduced, improves the efficiency of sequencing reaction.
The product of two pipe initial PCR amplifications is mixed into a pipe in equal volume, takes the PCR product mixture of 20ul, 2ul is added
FuPa Reagent, total volume is 22 μ L at this time, and whirlpool mixes well, and moment centrifuges 2 seconds, according to the program reset primer of table 6.
6 primer cleaning reaction program of table
PCR product can immediately using or be stored in -20 DEG C of refrigerators.
Five, linker fragment is obtained.
Connector is carried out to target fragment to connect to obtain belt lacing segment, is then purified using magnetic bead.
5.1 connectors react
Switch Solution are taken out, the target fragment after removing primer is placed in sample cell, whirlpool by room temperature mixing
It mixes well, moment centrifuges 2 seconds, and 2ul is taken to be added in connector reaction system as shown in table 7.
7 connector reaction system component list of table
Above-mentioned reaction system is put into PCR instrument, primer removing is carried out, response procedures are as shown in table 8.
8 connector response procedures table of table
Barcode is the single-stranded of synthesis, wherein the random sequence is made of 6~12 bases and at random positioned at close to single
The side that chain dissociative DNA 3 ' is held, the Adaptor sequences are arbitrary known array and are located remotely from single-stranded dissociative DNA 3 '
The side at end, two kinds of reagents are provided by Life Technologies.It is reacted by connecting, obtains each connection unique tags sequence
The sample of nucleic acid of row, sample can immediately using or be stored in -20 DEG C of refrigerators.
5.2 purifying, using Ampure XP Reagent magnetic beads：
1) 45 μ L (1.5 times of sample volumes) Ampure XP Reagent, mixing, incubation at room temperature are added into sample cell
5min；
2) sample cell is placed on magnetic frame, stands 2min, until solution is clarified completely in pipe；
3) supernatant is abandoned, keeps centrifuge tube on magnetic frame, it is mild to add (now with the current) washing of 150 μ L, 70% alcohol
Beads (gently rotates sample cell, fully wash)；
4) it repeats the above steps, fully removes alcohol, dry (dried on magnetic frame, should not overdrying).
5.3 measure the concentration of sample database
1) 1ul Qubit dsDNA HS Reagent and 199ul Qubit dsDNA HSbuffer are added to sample tube,
Whirlpool mixing 4s；
2) 3ul working solutions are abandoned to the sample cell interior suction for having added working solution, is added the DNA sample of 3ul, whirlpool mixing 4s,
Often the final volume of pipe is 200ul；
3) all pipes are protected from light incubation 2 minutes in room temperature；
4) exist" DNA " key is clicked in 3.0 main screens, then dsDNA High Sensitivity is selected to analyze mould
Formula；
5) sample tube is put intoIt in 3.0, closes the lid, clicks read；
6) Calculate Initial initial concentrations are selected, Volume of Sample Used are then selected:3ul, this
When the result that shows be sample initial concentration, unit ng/ul；
7) next sample is read：Sample in Qubit3.0 fluophotometers is taken out, new sample is put into, presses " Read
Next Ssmple”；
8) repeated sample is read, until all samples are run through.
Six, amplified library and purifying
6.1 amplified library
Since library construction is multi-PRC reaction, each primer pair has in amplified reaction with other primer competition
DNTPs, polymerase and other reagents of limitation, therefore exist and improved method and composition is needed, to allow selectivity to expand
Increase multiple target nucleic acid molecules in a group nucleic acid molecules, while avoiding the formation of non-specific amplification product and primer dimer,
Or minimize the degree of non-specific amplification product and primer dimer, reach more efficient, low concentration detection.The present invention
Organic reagent is added in the reaction system, organic reagent includes glycerine, olive oil, DMSO (dimethyl sulfoxide (DMSO)), it is preferable that in text
The organic reagent that library amplification is added is glycerine.
It is (black that 50 μ L Platinum PCR Super Mix High Fidelity are added into sample air-dried after purification
Color lid), the mixed liquor of first group of primer combination liquid of 3.5ul glycerine and 2ul and second group of primer combination liquid, it is mixed that magnetic bead is resuspended
It is even, 3min is placed on magnetic frame, is taken in supernatant to PCR pipe, and whirlpool mixing carries out PCR amplification, amplified reaction program such as 9 institute of table
Show.
9 amplified library response procedures table of table
Glycerine can improve library yield, be conducive to upper machine sequencing after amplification, the PCR product after amplification can use immediately or
It is stored in -20 DEG C of refrigerators.
6.2 libraries purify
Library carries out magnetic beads for purifying, using AMPure XP Reagent magnetic beads：
1) PCR product is transferred in 1.5ml centrifuge tubes, 25ul (0.5 times of sample volume) AMPure XP is added
Reagent fully blows and beats mixing, is incubated at room temperature 5min；
2) sample is placed on magnetic frame, 5min solution in managing is waited for clarify completely；
4) it careful Aspirate supernatant and is transferred in new 1.5ml centrifuge tubes；
5) the AMPure XPReagent magnetic of 60ul (1.2 times of original sample volumes) is added into the centrifuge tube of above-mentioned steps
Pearl blows and beats mixing, is incubated at room temperature 5min；
6) sample tube is placed on magnetic frame static 3min or is clarified to solution, abandon supernatant；
7) keep centrifuge tube on magnetic frame, mild 75% alcoholic solution for adding 150ul Fresh washs beads
(gently rotating sample cell, fully wash), rotating centrifugal pipe carefully removes alcohol；
8) cleaning that repeats the above steps is primary, fully removes alcohol, dries (dried on magnetic frame, should not overdrying), room temperature
Magnetic bead 5min is air-dried, it should not be over-drying；
9) sample cell is removed from magnetic frame, is added in 50 μ L Low TE solution to sample tube, whirlpool mixing；
10) sample tube being placed on magnetic frame, the supernatant of elution is transferred in new centrifuge tube by static 2min,
Sample concentration is detected with Qubit；
6.3 with3.0 measure the concentration of sample database
1) 1ul Qubit dsDNA HS Reagent and 199ul Qubit dsDNA HS are added to sample tube
Buffer, whirlpool mixing 4s；
2) 3ul working solutions are abandoned to the sample cell interior suction for having added working solution, is added the DNA sample of 3ul, whirlpool mixing 4s,
Often the final volume of pipe is 200ul；
3) all pipes are protected from light incubation 2 minutes in room temperature；
4) exist" DNA " key is clicked in 3.0 main screens, then dsDNA High Sensitivity is selected to analyze mould
Formula；
5) sample tube is put intoIt in 3.0, closes the lid, clicks read；
6) Calculate Initial initial concentrations are selected, Volume of Sample Used are then selected:3ul, this
When the result that shows be sample initial concentration, unit ng/ul；
7) next sample is read：Sample in Qubit3.0 fluophotometers is taken out, new sample is put into, presses " Read
Next Sample”；
8) repeated sample is read, until all samples are run through；
Library after purification directly can carry out upper machine sequencing by Ion Torrent platforms, after sequencing terminates, to obtaining
Sequencing data analyzed, by comparing the primer and site information of design, it is prominent to filter out gene on lung cancer related gene
Become and makes a variation.
The testing result of the present invention is consistent with clinic ARMS method testing results, and the distributed number of sequencing reaction sample is uniform.
The present invention has the advantages that lung cancer polygenic variation to detect flux height, high specificity, not easy to pollute, safe, detection knot
Fruit has preferable accuracy and repeatability, provides assistance in diagnosis to the clinical treatment of patients with lung cancer and suggesting effect.According to receipts
The pattern detection situation of collection, according to the SNP site that the result of negative sample removes, 5 patients with lung cancer are examined altogether on 10 genes
11 hot spot SNP sites are measured, and normal structure sample does not detect the hot spot SNP site.
In embodiment of the present invention, the genetic mutation is present in lung cancer but is not limited only to the lung cancer phase listed such as table 1
Correlation gene, there is also the related genes such as common gastric cancer, liver cancer, breast cancer, intestinal cancer, the cancer of the esophagus.In addition, it should also be understood that, having read this
After the content of invention, those skilled in the art can make various modifications or changes to the present invention, and such equivalent forms are equally fallen
In limited range of the present invention.Inventor obtains one group of library that can be used for Ion Proton through furtheing investigate and screening,
The library of structure can directly can the sequencing of above machine.
Polygenic variation includes at least two in mutation, copy number variation and inversion, can be comprehensively to patients with lung cancer
Treatment and prognosis make guide.
In order to reduce the requirement to tissue samples concentration and quality, improve the success rate of sequencing library structure, the present invention into
1) following trial of having gone uses the tissue extracting DNA sample of 1ng, 3ng, 5ng, 7ng, 9ng, according to 55 DEG C of common annealing temperature
Initial concentration amplification is carried out, uses 60 DEG C of annealing temperatures, the concentration for measuring library to be below 0.5ng/ul amplified library.To first
Begin to expand and the gradient experiment of 50 DEG C~65 DEG C of the annealing temperature of amplified library progress, the success rate in library are still very low；2) root
Primer pair is grouped according to software Design primers parameter, grouping is boundary, sense primer G/C content and downstream primer GC with 50%
It is first group of Primer composition that the average value of content, which is less than or equal to 50%, sense primer G/C content and downstream primer G/C content it is flat
It is second group of Primer composition that mean value, which is more than 50%, and 50 DEG C of annealing temperatures that first group of Primer composition uses carry out initial concentration
Amplification, amplified library use 60 DEG C of annealing temperatures, 60 DEG C of annealing temperatures that second group of Primer composition uses to carry out initial concentration
Amplification, amplified library use 60 DEG C of annealing temperatures, are not in charge of amplification relative to primary amplification, although the success rate in library is carried
Height but is below 50%；3) annealing temperature is to influence the more important factor of PCR specificity, and good annealing temperature can make primer
It is combined with template.Since template DNA is more complicated than primer, annealing temperature and time depend on length, the base group of primer
At, also template sequence composition and length.The present invention has considered the G/C content and annealing temperature parameter pair of primer and product
The influence of PCR reactions, devises to be in charge of and judges parameter R, according to judging parameter R to primer pair grouping, in primary amplification and library
The annealing temperature of amplification carries out 50 DEG C~65 DEG C of gradient test, selects the higher annealing temperature of library success rate, final preferred
The annealing temperature of first group of primer combination liquid primary amplification is 55 DEG C, and the annealing temperature of second group of primer combination liquid primary amplification is
65 DEG C, the mixed liquor of first group of primer combination liquid and second group of primer combination liquid is 62 DEG C in the annealing temperature of amplified library, text
Library structure success rate is significantly promoted, the case where especially suitable for library initial amount down to 1ng~10ng.
Above-described embodiment is only intended to clearly illustrate examples made by the present invention, and is not to embodiments of the present invention
Restriction.For those of ordinary skill in the art, other not similar shapes can also be made on the basis of the above description
The variation or variation of formula.It should be pointed out that for those of ordinary skill in the art, not departing from the invention design
Under the premise of, various modifications and improvements can be made, these are all within the scope of protection of the present invention.
Claims (9)
1. a kind of lung cancer polygenic variation library constructing method, which is characterized in that include the following steps：
A. according to the variable region of lung cancer related gene, design can detect the primer pair of these variable regions, the primer pair
5 ' end introduce deoxyinosine nucleotide base；
B. parameter W1, Tm1, W2 and Tm2 of each primer pair are obtained by primer-design software, to which each primer pair be calculated
Judge that parameter R, calculation formula are as follows：
R=0.1×[W1+Tm1/(Tm1+Tm2)]+0.9×[W2+Tm2/(Tm1+Tm2)]；
Wherein, W1 is the G/C content of amplified production, and Tm1 is the annealing temperature of amplified production, and W2 is sense primer G/C content under
The average value of primer G/C content is swum, Tm2 is the average value of sense primer annealing temperature and downstream primer annealing temperature,
It will determine that the value of parameter R is less than or equal to 1 primer pair, be classified as first group of primer combination liquid, will determine that the value of parameter R is big
In 1 primer pair, it is classified as second group of primer combination liquid；
C. by sample to be tested DNA extracting and purifyings；
D. first group of primer combination liquid is respectively adopted and second group of primer combination liquid carries out initial PCR amplification to sample after purification
Target fragment is obtained, primer is then removed, annealing temperature when liquid carries out initial PCR amplification is combined using first group of primer and is less than
Annealing temperature when liquid carries out initial PCR amplification is combined using second group of primer；
E. it carries out connector to the target fragment to connect to obtain belt lacing segment, then be purified；
F. first group of primer is used to combine the mixed liquor of liquid and second group of primer combination liquid to belt lacing segment progress after purification
Then Library PCR amplification is purified, sequencing library is obtained.
2. lung cancer polygenic variation library constructing method according to claim 1, it is characterised in that：The lung cancer dependency basis
Because include AKT1, ALK, BRAF, BRCA1, BRCA2, CDKN2A, C-met, DDR2, ERBB3, FGFR2, FGFR4, HER2,
KRAS, MAP2K1, mTOR, NRAS, NRF2, PIK3CA, PTCH1, RET, SMO, STK11, TSC1, TSC2, EGFR, TP53,
It is one or more in PTEN, FGFR1；Wherein AKT1, ALK, BRAF, BRCA1, BRCA2, CDKN2A, C-met, DDR2,
ERBB3, FGFR2, FGFR4, HER2, KRAS, MAP2K1, mTOR, NRAS, NRF2, PIK3CA, PTCH1, RET, SMO, STK11,
The variation sequencing type of TSC1, TSC2 are mutation sequencings, and the variation sequencing type of EGFR, TP53, PTEN are exon sequencings；C-
The variation sequencing type of met, HER2, FGFR1, PIK3CA are amplification sequencings.
3. lung cancer polygenic variation library constructing method according to claim 1, it is characterised in that：The primer pair is covered
The variable region of lid includes chr1：162754624-162754667、chr1：162754761-162754855、chr1：
162759815-162759873、chr1：162775671-162775740、chr1：162778592-162778698、chr14:
104770390-104770420、chr14:104772927-104772980、chr14:104773502-104773537、
chr14:104773920-104773953、chr14:104775128-104775143、chr14:104776719-
104776747、chr14:104780114-104780142、chr14:104792600-92637、chr2:29193886-
29193891、chr2:29196820-29196858、chr2:29197575-29197633、chr2:29214035-
29214042、chr2:29223520-29223528、chr2:29225500-29225518、chr2:29226922-
29226934、chr2:29296963-29296968、chr2:29318371-29318380、chr2:29328373-
29328388、chr2:29333767-29333795、chr2:29694861-29694888、chr2:29920019-
29920086、chr7:140734607-140734688、chr7:140753301-140753370、chr17：43082440-
43082543、chr17：43092005-43092100、chr17：43092907-43092953、chr17：43093990-
43094074、chr17：43106475-43106492、chr17：43115753-43115774、chr13：32326505-
32326575、chr13：32332322-32332386、chr13：32332536-32332638、chr13：32333139-
32333219、chr13：32336459-32336562、chr13：32337435-32337471、chr13：32363206-
32363316、chr13：32398684-32398766、chr9：21971186-21971208、chr9：21974764-
21974803、chr9：21968234-21968234、chr12：56085007-56085128、chr12：56086541-
56086555、chr12：56087577-56087640、chr12：56096505-56096539、chr12：56097153-
56097184、chr12：56101967-56102051、chr10：121479871-121479899、chr10：121483723-
121483769、chr10：121503794-121503854、chr10：121538612-121538640、chr10：
121551339-121551363、chr10：121564503-121564565、chr10：121593750-121593777、chr5：
177089639-17789673、chr5：177093269-177093311、chr5：177096144-177096163、chr5：
177097296-177097324、chr12：25227304-25227374、chr12：25245285-25245312、chr15：
66387410-66387421、chr15：66443280-66443310、chr15：66490556-66490572、chr1：
11117046-11117073、chr1：11130582-11130597、chr1：11109657-11109693、chr1：
11258571-11258588、chr1：114716116-11471652、chr2:177230956-177230973、chr2:
177231581-177231624、chr2:177231869-177231911、chr9:95446922-95446944、chr9:
95453505-95453539、chr9:95459693-95459726、chr9:95469086-95469107、chr9:
95508250-95508271、chr10:43100510-43100560、chr10:43111228-43111238、chr10:
43111342-43111412、chr10:43121969-43121991、chr7:129189158-129189225、chr7:
129210436-129210486、chr7:129212139-129212169、chr19:1207007-1207038、chr19:
1219351-1219462、chr19:1223130-1223160、chr9:132896605-132896654、chr9:
132902616-132902673、chr9:132904419-132904449、chr9:132905730-132905803、chr9:
132906751-132906779、chr9:132928836-132928863、chr16:2048689-2048723、chr16:
2050409-2050486、chr16:2061964-2062005、chr16:2070478-2070523、chr16:2071878-
2071911、chr16:2084534-2084653、chr16:2086217-2086296、chr3:179198915-179198966、
chr3:179199803-179199860、chr3:179203618-179203697、chr3:179204553-179204586、
chr3:179210214-179210276、chr17:39707104-39707141、chr17:39710358-39710389、
chr17:39715774-39715862、chr17:39724825-39724865、chr17:39726571-39726598、
chr17:39727871-39727981、chr7:116731688-116731707、chr7:116740046-116740083、
chr7:116771552-116771574、chr7:116782003-116782070、chr7:116795915-116795945、
chr7:116775003-116775102、chr8:38413973-38414170、chr8:38415881-38415915、chr8:
38417348-38417370、chr8:38429801-38429814、chr8:38457373-38457422、chr7:
55173921-55174039、chr7:55174726-55174810、chr7:55181301-55181337、chr7:
55181365-55181457、chr7:55191729-55191869、chr17:7669616-7669662、chr17:7673736-
7673788、chr17:7673736-7673788、chr17:7673736-7673831、chr17:7674183-7674253、
chr17:7675053-7675108、chr17:7676214-7676245、chr10:87933035-87933086、chr10:
87933124-87933183、chr10:One or more of 87960993-87961048.
4. lung cancer polygenic variation library constructing method according to claim 1, it is characterised in that：In the step F
Mixed liquor is that first group of primer combination liquid and second group of primer combination liquid are mixed according to volume ratio for 1 ﹕ 1.
5. lung cancer polygenic variation library constructing method according to any one of claim 1 to 4, it is characterised in that：Institute
When stating initial PCR amplification in step D, dimethyl sulfoxide (DMSO) is added in the reaction system, the volume that dimethyl sulfoxide (DMSO) is added accounts for reaction
The 3%~7% of system volume.
6. lung cancer polygenic variation library constructing method according to any one of claim 1 to 4, it is characterised in that：Institute
When stating step F Chinese library PCR amplifications, glycerine is added in the reaction system, the volume that glycerine is added accounts for the 5% of reaction system volume
~8%.
7. lung cancer polygenic variation library constructing method according to any one of claim 1 to 4, it is characterised in that：Institute
Stating experiment condition of first group of primer mixed liquor in initial PCR amplification in step D is：99 DEG C, 2min, 1 cycles；99 DEG C,
15s, 55 DEG C, 20s, 72 DEG C, 15 cycles of 30s；
Experiment condition of the second group of primer combination liquid in initial PCR amplification is in the step D：99 DEG C, 2min, 1 cycles；
99 DEG C, 15s, 65 DEG C, 20s, 72 DEG C, 30s, 15 cycles.
8. lung cancer polygenic variation library constructing method according to any one of claim 1 to 4, it is characterised in that：Institute
State the mixed liquor of first group of primer combination liquid and first group of primer combination liquid in step F is in the experiment condition of amplified library：98
DEG C, 2min, 1 cycle；99 DEG C, 15s, 62 DEG C, 1min, 5 cycles.
9. a kind of sequencing library using constructed by lung cancer polygenic variation library constructing method as described in claim 1.
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| CN107619867A (en) * | 2017-10-18 | 2018-01-23 | 广州漫瑞生物信息技术有限公司 | For detecting the combined sequence and probe of lung cancer several genes mutation type simultaneously |
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| CN108753924A (en) * | 2018-06-08 | 2018-11-06 | 安徽鼎晶生物科技有限公司 | A kind of lung cancer high-throughput sequencing library and its construction method and application |
| CN110029103B (en) * | 2019-04-30 | 2022-12-27 | 上海思路迪生物医学科技有限公司 | Automatic kit for constructing BRCA1/2 gene variation detection library |
| WO2020228009A1 (en) * | 2019-05-16 | 2020-11-19 | 北京市肿瘤防治研究所 | Method for quantitatively detecting deletion of human cdkn2a gene copy, primers and use thereof |
| CN115772565B (en) * | 2021-09-08 | 2023-09-05 | 广州市基准医疗有限责任公司 | Methylation site for auxiliary detection of lung cancer somatic EGFR gene mutation and application thereof |
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