CN107119331A - The construction method of tumour radiotherapy virulent gene mutated library - Google Patents
The construction method of tumour radiotherapy virulent gene mutated library Download PDFInfo
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Abstract
The invention discloses a kind of construction method of tumour radiotherapy virulent gene mutated library, it is characterised in that：Cover on related 42 genes of human tumor radiotherapy toxicity 61 kinds of hereditary variation sites altogether.The construction method of the present invention carries out single tube for multiple target sequences, it is rapidly completed the structure in library, whole library construction process only needs 2~3 hours, need only to manufal operation time 45 minutes, it can receive to predict whether can occur larger toxic side effect before tumour radiotherapy for clinically patient at present with highly effective solution, and need to detect polygenes, Mutiple Targets this difficult point, and it is with low cost.
Description
Technical field
The present invention relates to a kind of structure of the tumour radiotherapy virulent gene mutated library detected for high-flux sequence
Method.
Background technology
Malignant tumour is the main public health problem of China and the whole world.The 1/4 of whole causes of the death is accounted in China's tumor mortality.
Radiotherapy has therapeutic action to Several Kinds of Malignancy, is one of conventional treatment method.Increasing patient is by putting
Penetrate treatment and alleviate slight illness, extend life.About 70% malignant tumor patient, difference is needed in a certain period of its course of disease
The radiotherapy of purpose is participated in, including radical radiation therapy, adjuvant radiotherapy or Palliative radiotherapy.The machine of radiotherapy killing tumor cell
System is to act on organism generation by radioactive ray to stimulate electronics to draw ionization damage DNA molecular, or passes through ray and biological tissue
The effect of interior hydrone produces free radical damage dna molecule, when the repair ability that damage exceeds cell is to cause cell death
And disorganization.
Perfect tumor therapeuticing method is preferably killed effectively and had no toxic side effect to tumor tissues.With science and technology
Development, people, which gradually improve traditional radiation therapy technology, which has developed, can more precisely kill the treatment method of tumor tissues, from
And reduce normal structure suffered dose of radiation in the treatment.But in clinical practice oncotherapy, radiotherapy is disliked in killing
While property tumour cell, the normal structure and organ of tumor vicinity are also inevitably by radioactive damage, so as to produce
Radiation treatment toxic side effect.Cardiac toxic is most to threaten one of Esophageal carcinoma of patient vitals, inadiation induction heart disease,
Belong to late complication, occur typically after radiotherapy 10-20；Show as pericarditis, cardiomyopathy, valvulopathy, conduct it is different
Normal and coronary artery stenosis.In HD long term survival patients, the patient for having 2%-5% ultimately succumb to heart injury disease rather than
Primary disease, myocardial infarction is then the major causes of death of long term survival patient after these tomor rejections.Poison is secondary caused by radiotherapy
Act on and different degrees of pain is brought to patient, severe patient has an effect on being smoothed out for treatment.
It is predicted that caused by the individual difference of 80% pair of clinical treatment reaction is the individual factors for having patient related, determining this
A little factors can predict the risk of the serious toxicity of patient evolution.Therefore the side reaction that identification individual patients are obtained from radiotherapy
Neurological susceptibility is an important prerequisite of the individual chemoradiotherapy of tumour.By finding the risk related to Patients Treated by Radiotherapy toxic side effect
The factor, assesses the expression of patient's correlation factor so as to predict the radiotherapy toxic side effect neurological susceptibility of patient, can be resistance to normal tissue
Expanded treatment window by increasing tumor dosimetry by the patient of radiotherapy；Another aspect normal tissue toxicity has
The patient of excessive risk may change therapeutic scheme, change operation into or chemotherapy intervention improves symptom etc., so as to be provided effectively for patient
And the small therapeutic scheme of toxicity, improve the Survival of patient.
The content of the invention
For above shortcomings in the prior art, the invention provides a kind of tumour detected for high-flux sequence
The construction method of radiotherapy virulent gene mutated library.
In order to solve the above-mentioned technical problem, present invention employs following technical scheme：
The construction method of tumour radiotherapy virulent gene mutated library, covering human gene XRCC1, TNFa, TXNRD2,
ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、
MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、
61 kinds of heredity altogether on NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2,5q31, HSPB1 and TXN
Property variant sites, specifically include following steps：
(1) for target gene XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3,
LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、
IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、
Basic amplimer group is designed on MYO3B, TGFBR2,5q31, HSPB1 and TXN, the forward primer of the basic amplimer group and
5 ' ends of reverse primer are provided with 2~5 extra T, and first T close to 3 ' ends of 2~5 T has PNA modifications, together
When the basic amplimer group Tm values difference be no more than 1 DEG C；
(2) template, above-mentioned basic amplimer group are placed in the PCR reaction systems containing RingCap-Taq enzymes
Row amplification, obtains amplified production after purification；By amplified production, multipair first asymmetric linking probe, universal primer and above-mentioned
RingCap-Taq enzymes are mixed into performing PCR, and library production is obtained after purification；
(3) library production is constituted into the genetic mutation library for high-flux sequence；
Above-mentioned RingCap-Taq enzymes are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA.
As a preferred embodiment of the present invention, the basic amplimer group includes FL-XRCC1-1-F, FL-XRCC1-
1-R、FL-TNFa-1-F、FL-TNFa-1-R、FL-TXNRD2-1-F、FL-TXNRD2-1-R、FL-ERCC2-1-F、FL-
ERCC2-1-R、FL-TGFB1-1-F、FL-TGFB1-1-R、FL-VEGF-1-F、FL-VEGF-1-R、FL-DNMT1-1-F、FL-
DNMT1-1-R、FL-XRCC1-2-F、FL-XRCC1-2-R、FL-TP53-1-F、FL-TP53-1-R、FL-XRCC3-1-F、FL-
XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、
FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-
ATM-1-R、FL-XRCC1-3-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、
FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、
FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC1-3-R、FL-NOS2-1-F、FL-
NOS2-1-R、FL-TGFB1-2-F、FL-TGFB1-2-R、FL-APEX1-1-F、FL-APEX1-1-R、FL-VEGF-2-F、FL-
VEGF-2-R、FL-XRCC1-4-F、FL-XRCC1-4-R、FL-MLH1-1-F、FL-MLH1-1-R、FL-IL8-1-F、FL-IL8-
1-R、FL-CD44-1-F、FL-CD44-1-R、FL-LIG4-1-F、FL-LIG4-1-R、FL-IL1A-1-F、FL-IL1A-1-R、
FL-TNF-1-F、FL-TNF-1-R、FL-CYP2C8-1-F、FL-CYP2C8-1-R、FL-IL1A-2-F、FL-IL1A-2-R、FL-
IL4-1-F、FL-IL4-1-R、FL-IL4-2-F、FL-IL4-2-R、FL-NEIL1-1-F、FL-NEIL1-1-R、FL-NBN-1-
F、FL-NBN-1-R、FL-APC-1-F、FL-APC-1-R、FL-NFKBIA-1-F、FL-NFKBIA-1-R、FL-DEAD-1-F、
FL-DEAD-1-R、FL-ATM-2-F、FL-ATM-2-R、FL-IL8-2-F、FL-IL8-2-R、FL-ATM-3-F、FL-ATM-3-
R、FL-tPA-1-F、FL-tPA-1-R、FL-TGFB1-3-F、FL-TGFB1-3-R、FL-RAD51-1-F、FL-RAD51-1-R、
FL-NFE2L2-1-F、FL-NFE2L2-1-R、FL-GSTA1-1-F、FL-GSTA1-1-R、FL-MGMT-1-F、FL-MGMT-1-
R、FL-ATM-4-F、FL-ATM-4-R、FL-TDG-1-F、FL-TDG-1-R、FL-TDG-2-F、FL-TDG-2-R、FL-GSK3B-
1-F、FL-GSK3B-1-R、FL-NOS2-2-F、FL-NOS2-2-R、FL-XRCC1-5-F、FL-XRCC1-5-R、FL-FSHR-1-
F、FL-FSHR-1-R、FL-MYO3B-1-F、FL-MYO3B-1-R、FL-TGFBR2-1-F、FL-TGFBR2-1-R、FL-
TGFBR2-2-F、FL-TGFBR2-2-R、FL-5q31-1-F、FL-5q31-1-R、FL-LIN28B-2-F、FL-LIN28B-2-R、
FL-HSPB1-1-F, FL-HSPB1-1-R, FL-NBN-2-F, FL-NBN-2-R, FL-TXN-1-F and FL-TXN-1-R.
It is used as another preferred scheme of the present invention：XRCC1 gene primer titles：FL-XRCC1-1-F, sequence information
TTTTCTGCTGCAGGACACGACA；FL-XRCC1-1-R, sequence information TTTTCGCGCTTGCGCACTTTA；
TNFa gene primer titles：FL-TNFa-1-F, sequence information TTTTCCCTCCCAGTTCTAGTTCTAT；FL-
TNFa-1-R, sequence information TTTTCTTCTGGGCCACTGACTGATT；
TXNRD2 gene primer titles：FL-TXNRD2-1-F, sequence information TTTTCACATTGTCGTAGTCCATCAGA；
FL-TXNRD2-1-R, sequence information TTTTGCAAAGGGTGATGGCATAGG；
ERCC2 gene primer titles：FL-ERCC2-1-F, sequence information TTTTAAGACTCAGGAGTCACCAGGA；FL-
ERCC2-1-R, sequence information TTTTTCTGTTCTCTGCAGGAGGATC；
TGFB1 gene primer titles：FL-TGFB1-1-F, sequence information TTTTGTCAGGCTGGGAAACAAGGTA；FL-
TGFB1-1-R, sequence information TTTTGGTGCTCAGTAAAGGAGAGCAA；
VEGF gene primer titles：FL-VEGF-1-F, sequence information TTTTCCCAAATCACTGTGGATTTTG；FL-
VEGF-1-R, sequence information TTTTCCCAAAAGCAGGTCACTCACT；
DNMT1 gene primer titles：FL-DNMT1-1-F, sequence information TTTTCCCAAACATAATCCCGGACTAT；FL-
DNMT1-1-R, sequence information TTTTTAAGCATGTGCTTTGTTTCCTGT；
XRCC1 gene primer titles：FL-XRCC1-2-F, sequence information TTTTCTGGGACCACCTGTGTTCT；FL-
XRCC1-2-R, sequence information TTTTCCGCATCGTGCGTAAGGA；
TP53 gene primer titles：FL-TP53-1-F, sequence information TTTTGGTTTTCTGGGAAGGGACAGA；FL-
TP53-1-R, sequence information TTTTCGGACGATATTGAACAATGGTTC；
XRCC3 gene primer titles：FL-XRCC3-1-F, sequence information TTTTCGCATCCTGGCTAAAAATACGA；FL-
XRCC3-1-R, sequence information TTTTATTCCGCTGTGAATTTGACAG；
LIN28B gene primer titles：FL-LIN28B-1-F, sequence information
TTTTTGAATTAAAACATGTAGCTGCTGAAG；FL-LIN28B-1-R, sequence information
TTTTAAGAAACCTCTTTAACTAGGCATCA；
LIN28B gene primer titles：FL-LIN28B-1-F, sequence information
TTTTTGAATTAAAACATGTAGCTGCTGAAG；FL-LIN28B-1-R, sequence information
TTTTAAGAAACCTCTTTAACTAGGCATCA；
XRCC3 gene primer titles：FL-XRCC3-2-F, sequence information TTTTAGTGTCCACTGACGGATAACAGA；
FL-XRCC3-2-R, sequence information TTTTCCTAATCAGCTGTCAAGGGTGAT；
Mthfr gene Primer：FL-MTHFR-1-F, sequence information TTTTCACTCCAGCATCACTCACTTTG；FL-
MTHFR-1-R, sequence information TTTTGGGAGCTGAAGGACTACTACCT；
PON1 gene primer titles：FL-PON1-1-F, sequence information TTTTATACTTGCCATCGGGTGAAATGT；FL-
PON1-1-R, sequence information TTTTGCACTTTTATGGCACAAATGATCAC；
GSTP1 gene primer titles：FL-GSTP1-1-F, sequence information TTTTAGTGACTGTGTGTTGATCAGGC；FL-
GSTP1-1-R, sequence information TTTTAGATGCTCACATAGTTGGTGTAGATG；
ATM gene primer titles：FL-ATM-1-F, sequence information TTTTACTCATTTTTACTCAAACTATTGGG；FL-
ATM-1-R, sequence information TTTTCGAAGACAGCTGGTGAAAAATCC；
XRCC1 gene primer titles：FL-XRCC1-3-F, sequence information TTTTCTACCACACCCTGAAGGATCTTC；
FL-XRCC1-3-R, sequence information TTTTCTAATCTACTCTTTGTCTTCTCCAGT；
NOS2 gene primer titles：FL-NOS2-1-F, sequence information TTTTGGCTAGGAGTAGGACAACGGAA；FL-
NOS2-1-R, sequence information TTTTTGGCCAGGTTTCCAGAAGAAAG；
TGFB1 gene primer titles：FL-TGFB1-2-F, sequence information TTTTGTAACCATCATGGGCCTTGTC；FL-
TGFB1-2-R, sequence information TTTTTGTTCTGAGGACATGGGCAAA；
APEX1 gene primer titles：FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT；
FL-APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC；
APEX1 gene primer titles：FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT；
FL-APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC；
VEGF gene primer titles：FL-VEGF-2-F, sequence information TTTTCACACCATCACCATCGACAGAA；FL-
VEGF-2-R, sequence information TTTTGCAAGAAAAATAAAATGGCGAATC；
XRCC1 gene primer titles：FL-XRCC1-4-F, sequence information TTTTCGAGTCTAGGTCTCAACCCTAC；FL-
XRCC1-4-R, sequence information TTTTCGGGACCTTAGAAGGTGACAGT；
MLH1 gene primer titles：FL-MLH1-1-F, sequence information TTTTCAACCCACAGAGTTGAGAAATTTG；FL-
MLH1-1-R, sequence information TTTTAAGAGCCAAGGAAACGTCTAGATG；
IL8 gene primer titles：FL-IL8-1-F, sequence information TTTTTGTTCTAACACCTGCCACTCTAGT；FL-
IL8-1-R, sequence information TTTTCGGAGTATGACGAAAGTTTTCTTTGA；
CD44 gene primer titles：FL-CD44-1-F, sequence information TTTTGGTCCCTGGGAGGAAATTTGAA；FL-
CD44-1-R, sequence information TTTTGTGTCTCCCAGAAGCATCTGAAAA；
LIG4 gene primer titles：FL-LIG4-1-F, sequence information
TTTTAAGAATCTAAAAATTCCCTGAAGTGTCT；FL-LIG4-1-R, sequence information
TTTTTGCTTTACTAGTTAAACGAGAAGATTCAT；
IL1A gene primer titles：FL-IL1A-1-F, sequence information TTTTCAGCCGTGAGGTACTGATCATT；FL-
IL1A-1-R, sequence information TTTTCATTAATCTGCACTTGTGATCATGGTT；
Tnf gene Primer：FL-TNF-1-F, sequence information TTTTGATGTGACCACAGCAATGGGTA；FL-TNF-
1-R, sequence information TTTTCCCAGTGTGTGGCCATATCTTC；
CYP2C8 gene primer titles：FL-CYP2C8-1-F, sequence information
TTTTTCAATTGGGAACAACAGAGTTAACTCC；FL-CYP2C8-1-R, sequence information
TTTTTGGAATTAGTTGGAATTTACATG；
IL1A gene primer titles：FL-IL1A-2-F, sequence information TTTTAGAAGCCAGTGGCTAAGTTTGG；FL-
IL1A-2-R, sequence information TTTTTTCATTTGCTAAGAGTCTGGTGTTCT；
IL4 gene primer titles：FL-IL4-1-F, sequence information TTTTCCCAAGTGACTGACAATCTGGT；FL-IL4-
1-R, sequence information TTTTCCCATTAATAGGTGTCGATTTGCAGT；FL-IL4-2-F, sequence information
TTTTCCCAAACTAGGCCTCACCTGATA；FL-IL4-2-R, sequence information TTTTGTACAGGTGGCATCTTGGAAACT；
NEIL1 gene primer titles：FL-NEIL1-1-F, sequence information TTTTGGCTTCTCAACTCATGGTCTCT；FL-
NEIL1-1-R, sequence information TTTTGATGGCTTCCCAGGTATTTGGT；
NBN gene primer titles：FL-NBN-1-F, sequence information TTTTATCCTGAAACAAGCATTAAAGAGGGA；FL-
NBN-1-R, sequence information TTTTCGTCCAATTGTAAAGCCAGAATATTTT；
Apc gene Primer：FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT；
FL-APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT；
Apc gene Primer：FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT；
FL-APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT；
NFKBIA gene primer titles：FL-NFKBIA-1-F, sequence information
TTTTAGTGTGCAGTGTGGATATAAGTACAC；FL-NFKBIA-1-R, sequence information
TTTTTTTCAGCTGCCCTATGATGACTG；
DEAD gene primer titles：FL-DEAD-1-F, sequence information TTTTTCCTTGCTTCCAGTTTTTCCACT；FL-
DEAD-1-R, sequence information TTTTGGACTTTATCCACTGAAATGTGATA；
ATM gene primer titles：FL-ATM-2-F, sequence information TTTTTGCGTGGCTAACGGAGAAAA；FL-ATM-2-
R, sequence information TTTTGGGTCGCACACGACTGAATT；
IL8 gene primer titles：FL-IL8-2-F, sequence information TTTTGCCTACTATAAATAACACTGTGGTA；FL-
IL8-2-R, sequence information TTTTCCCTTGACCTCAGTTAGTTCTTTGTT；
ATM gene primer titles：FL-ATM-3-F, sequence information TTTTAGGGAAATCATAAAGTACGTGAGTCT；FL-
ATM-3-R, sequence information TTTTATGCTTTCTATTTCTCAGCAAAAACA；
TPA gene primer titles：FL-tPA-1-F, sequence information TTTTTAACACTGACAATAACCAAAACCAAG；FL-
TPA-1-R, sequence information TTTTCCCAGGTCTGAGTGATCTCATTG；
TGFB1 gene primer titles：FL-TGFB1-3-F, sequence information TTTTGTAGCCACAGCAGCGGTAGC；FL-
TGFB1-3-R, sequence information TTTTTTCCCTCGAGGCCCTCCTAC；
RAD51 gene primer titles：FL-RAD51-1-F, sequence information TTTTGCAACTCATCTGGGTTGTGC；FL-
RAD51-1-R, sequence information TTTTCTCACACACTCACCTCGGTC；
NFE2L2 gene primer titles：FL-NFE2L2-1-F, sequence information TTTTGGGCTAAAGATTTGGACCCAG；
FL-NFE2L2-1-R, sequence information TTTTGAATGGAGACACGTGGGAGTT；
GSTA1 gene primer titles：FL-GSTA1-1-F, sequence information
TTTTATAAGATCAGTACTTACTTTGTTAAA；FL-GSTA1-1-R, sequence information
TTTTGAGTGGCTTTTCCCTAACTTGACT；
Mgmt gene Primer：FL-MGMT-1-F, sequence information TTTTCCCATAGTTCAGTTCTCTGTGTAGG；
FL-MGMT-1-R, sequence information TTTTCTAGCACGAGGTGGGCAGAGT；
ATM gene primer titles：FL-ATM-4-F, sequence information TTTTTCTGTAATCTATAGCAGAGTAAAGCG；FL-
ATM-4-R, sequence information TTTTAAAAACAAAAAGAGCTGACTACCCA；
TDG gene primer titles：FL-TDG-1-F, sequence information TTTTCCCACATTGTTTCCTTTGAACAGA；FL-
TDG-1-R, sequence information TTTTAGTCTATGGCATTCTGAAACAGCAA；FL-TDG-2-F, sequence information
TTTTGTCTGAAGACACTTTTGATGATCAAG；FL-TDG-2-R, sequence information
TTTTCTCAGGACGTAAAGCAGTTTCTCA；
GSK3B gene primer titles：FL-GSK3B-1-F, sequence information
TTTTGTTTGCACAACTTCGTGAATCTACTC；FL-GSK3B-1-R, sequence information
TTTTTGACCTTAAGTGCATGGACATTGG；
NOS2 gene primer titles：FL-NOS2-2-F, sequence information TTTTCGCCTCTGACAGATACACTCAG；FL-
NOS2-2-R, sequence information TTTTCAATCTGCAATAACGCAATAGTGCAA；
XRCC1 gene primer titles：FL-XRCC1-5-F, sequence information TTTTCCAGGTCACTTTTGGATTCTGGT；
FL-XRCC1-5-R, sequence information TTTTCCTCATGGTGTGTGTATCAGCA；
FSHR gene primer titles：FL-FSHR-1-F, sequence information TTTTCCGAGAAATCCAGAAATCTAGACTTA；
FL-FSHR-1-R, sequence information TTTTTCTACCCAGTGTATCATTCTGCTTACT；
MYO3B gene primer titles：FL-MYO3B-1-F, sequence information
TTTTCATGATCCTTTATACTGCCACCAGAA；FL-MYO3B-1-R, sequence information
TTTTTGGCAAGTTAATGTCCAAGATGGAATT；
TGFBR2 gene primer titles：FL-TGFBR2-1-F, sequence information TTTTTCTGAATGCCCAGAAGACCTCT；
FL-TGFBR2-1-R, sequence information TTTTGGGAAGCATGGAATATGACAAGAGTC；FL-TGFBR2-2-F, sequence information
TTTTCCACATCCATTTGGTGCTTTTAATTGA；FL-TGFBR2-2-R, sequence information
TTTTCCAGTCGATAACTAAGAAGAAGCTACA；
5q31 gene primer titles：FL-5q31-1-F, sequence information TTTTTTGATAACAGCAGAAGATAGAAGAGTT；
FL-5q31-1-R, sequence information TTTTCTGTATTCCCAAGACAAAGCCTCA；
LIN28B gene primer titles：FL-LIN28B-2-F, sequence information TTTTCAATTGTGTGAAGCCAGAGCAT；
FL-LIN28B-2-R, sequence information TTTTACATTCCTACGACATGGAGACAAATG；
HSPB1 gene primer titles：FL-HSPB1-1-F, sequence information TTTTGCAATGCATGACTGTTACCATCATT；
FL-HSPB1-1-R, sequence information TTTTGCAATCTAGTAATCTGGTGTGGTTGTTAG；
NBN gene primer titles：FL-NBN-2-F, sequence information TTTTATTATTTCAGCCAGATGGCAGACT；FL-
NBN-2-R, sequence information TTTTTCTGTAAAATAAGAATAGCAAAGGCAGT；
TXN gene primer titles：FL-TXN-1-F, sequence information TTTTACATACCCAAGCTTCTTAAATGGAAACT；
FL-TXN-1-R, sequence information TTTTCCTTATTTGAAATCCCAGCCTCAGAATT.
As another preferred scheme of the present invention, in step (2), enriched multiple PCR reaction system is：
1 × PCR buffer solutions
Scheme as a further improvement on the present invention, the kit of the construction method, including
One DNA is enriched with reaction component, is made up of the first amplimer group；
One RingCap-Taq enzymes, are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA；
One pipe positive quality control product；
With the negative quality-control product of a pipe.
Compared with prior art, the invention has the advantages that：
1st, construction method of the invention carries out single tube for multiple target sequences, is rapidly completed the structure in library, whole library
Building process only needs 2~3 hours, needs only to manufal operation time 45 minutes, can be very with reference to high-flux sequence platform
It is effective to solve whether occur relatively toxic for clinically predicting that patient receives tumour radiotherapy at present, and need to many bases
Cause, Mutiple Targets detect this difficult point, and with low cost.
2nd, the library sequence prepared by construction method of the invention by current high-flux sequence system identification and can be examined
Survey, so as to realize that library construction carries out the application of the detection of nucleotide sequence, the detection of nucleic acids can be applied to current a variety of high fluxs
Microarray dataset, genetic chip platform, hybridization check platform, are shown in Fig. 1.
3rd, construction method of the invention is applied to the DNA obtained from peripheral blood sample, sees Fig. 2, Fig. 3.
Brief description of the drawings
Fig. 1 is that the nucleic acid library that embodiments of the invention are built carries out high-flux sequence total data figure；
Fig. 2 is the detection homogeneity result figure that embodiments of the invention detect the mutation of tumour radiotherapy virulent gene；
Fig. 3 is the detection mutation result that embodiments of the invention detect the mutation of tumour radiotherapy virulent gene.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
The construction method of oncotherapy cardiac toxic gene mutation library, covering human gene XRCC1, TNFa, TXNRD2,
ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、
MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、
61 kinds of heredity altogether on NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2,5q31, HSPB1 and TXN
Property variant sites.
Specifically include following steps：
(1) for target gene XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3,
LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、
IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、
MYO3B, TGFBR2,5q31, HSPB1 and TXN design basic amplimer group, the forward primer of the basic amplimer group and anti-
To 5 ' ends of primer provided with 2~5 extra T, and first T close to 3 ' ends of 2~5 T has PNA modifications, simultaneously
The Tm values difference of the basic amplimer group is no more than 1 DEG C；The amplimer group includes FL-XRCC1-1-F, FL-XRCC1-
1-R、FL-TNFa-1-F、FL-TNFa-1-R、FL-TXNRD2-1-F、FL-TXNRD2-1-R、FL-ERCC2-1-F、FL-
ERCC2-1-R、FL-TGFB1-1-F、FL-TGFB1-1-R、FL-VEGF-1-F、FL-VEGF-1-R、FL-DNMT1-1-F、FL-
DNMT1-1-R、FL-XRCC1-2-F、FL-XRCC1-2-R、FL-TP53-1-F、FL-TP53-1-R、FL-XRCC3-1-F、FL-
XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、
FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-
ATM-1-R、FL-XRCC1-3-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、
FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、
FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC1-3-R、FL-NOS2-1-F、FL-
NOS2-1-R、FL-TGFB1-2-F、FL-TGFB1-2-R、FL-APEX1-1-F、FL-APEX1-1-R、FL-VEGF-2-F、FL-
VEGF-2-R、FL-XRCC1-4-F、FL-XRCC1-4-R、FL-MLH1-1-F、FL-MLH1-1-R、FL-IL8-1-F、FL-IL8-
1-R、FL-CD44-1-F、FL-CD44-1-R、FL-LIG4-1-F、FL-LIG4-1-R、FL-IL1A-1-F、FL-IL1A-1-R、
FL-TNF-1-F、FL-TNF-1-R、FL-CYP2C8-1-F、FL-CYP2C8-1-R、FL-IL1A-2-F、FL-IL1A-2-R、FL-
IL4-1-F、FL-IL4-1-R、FL-IL4-2-F、FL-IL4-2-R、FL-NEIL1-1-F、FL-NEIL1-1-R、FL-NBN-1-
F、FL-NBN-1-R、FL-APC-1-F、FL-APC-1-R、FL-NFKBIA-1-F、FL-NFKBIA-1-R、FL-DEAD-1-F、
FL-DEAD-1-R、FL-ATM-2-F、FL-ATM-2-R、FL-IL8-2-F、FL-IL8-2-R、FL-ATM-3-F、FL-ATM-3-
R、FL-tPA-1-F、FL-tPA-1-R、FL-TGFB1-3-F、FL-TGFB1-3-R、FL-RAD51-1-F、FL-RAD51-1-R、
FL-NFE2L2-1-F、FL-NFE2L2-1-R、FL-GSTA1-1-F、FL-GSTA1-1-R、FL-MGMT-1-F、FL-MGMT-1-
R、FL-ATM-4-F、FL-ATM-4-R、FL-TDG-1-F、FL-TDG-1-R、FL-TDG-2-F、FL-TDG-2-R、FL-GSK3B-
1-F、FL-GSK3B-1-R、FL-NOS2-2-F、FL-NOS2-2-R、FL-XRCC1-5-F、FL-XRCC1-5-R、FL-FSHR-1-
F、FL-FSHR-1-R、FL-MYO3B-1-F、FL-MYO3B-1-R、FL-TGFBR2-1-F、FL-TGFBR2-1-R、FL-
TGFBR2-2-F、FL-TGFBR2-2-R、FL-5q31-1-F、FL-5q31-1-R、FL-LIN28B-2-F、FL-LIN28B-2-R、
FL-HSPB1-1-F, FL-HSPB1-1-R, FL-NBN-2-F, FL-NBN-2-R, FL-TXN-1-F and FL-TXN-1-R, its sequence
Successively as shown in SEQ 01~SEQ of ID ID 122；
(2) template, above-mentioned basic amplimer group are placed in the PCR reaction systems containing RingCap-Taq enzymes
Row amplification, obtains amplified production after purification；By amplified production, multipair first asymmetric linking probe, universal primer and above-mentioned
RingCap-Taq enzymes are mixed into performing PCR, and library production is obtained after purification；
(3) library production is constituted into the tumour radiotherapy virulent gene mutated library for high-flux sequence；
(4) machine testing on library is carried out by Ion torrent PGM high-flux sequences instrument, obtains target sequence information,
Data message is carried out by VC softwares and compares analysis, sample mutation status are obtained.
Above-mentioned template from the sample scope of application include periphery blood specimen.
Peripheral blood sample, genomic DNA is extracted using Qiagen companies peripheral blood DNA extracts kit, and specific steps are pressed
Kit operating instruction.Peripheral blood is extracted every time is no less than 1000 μ l.Carried DNA be dissolved in Tris-HCl (10mmol/L,
PH8.0), quality is extracted through UV spectrophotometer measuring, and determines concentration, with Tris-HCl solution (10mmol/L, pH
8.0) template that adjustment DNA concentration is expanded to 2ng/ μ l as PCR.
Kit based on above-mentioned construction method includes：
One DNA is enriched with reaction component, is made up of amplimer group, and the formula of the DNA enrichment reaction components per person-portion is as follows
Shown in table：
One RingCap-Taq enzymes, are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA, and ratio is 0.8~1.2:
0.8~1.2:0.8~1.2, preferred proportion 1:1:1；
One negative quality-control product, specially seedless sour water；
With a positive quality control product, specifically mixed by 10 Positive mutants plasmid sequence wild type gene group DNA, concentration
For 2ng/ μ L.
Mentioned reagent box is tested with foregoing construction method, to analyze the detection tumour radiotherapy poison of the present invention
The method of property gene mutation.Faced respectively with the 20 parts of whole blood samples of clinical breast cancer radiation therapy process without serious toxicity, 20 parts
There is the whole blood sample of serious toxicity in bed radiotherapy of Breast Cancer process：
The template amount (testing sample, positive quality control product and negative quality-control product) of the PCR reaction systems is 5uL, remaining group
Divide as shown in the table：
PCR amplification programs set such as following table：
The purifying of amplified production obtained by above-mentioned PCR reaction systems is specific as follows：
Take out Agencourt AMPure XP reagents and be placed in room temperature, while magnetic bead is broken up, while preparing fresh 70%
Ethanol (the μ L nuclease-free waters of 230 μ L absolute ethyl alcohols+100), it is necessary to be Fresh.
First round purification step
(1) Agencourt of 12.5 μ L (0.5x sample volumes) is separately added into each μ L products of example reaction pipe 25
AMPure XP reagents, draw piping and druming 5 times up and down, mix DNA is resuspended completely；
(2) it is incubated at room temperature 5 minutes；
(3) it is placed on above magnetic frame, is incubated 5 minutes, until solution clarification；
(4) carefully Aspirate supernatant is placed in new centrifuge tube, should not disturb magnetic bead；Note：Contain amplification in supernatant
Product should not be abandoned.
Second wheel purification step
(1) the Agencourt AMPure of 30 μ L (1.2x sample volumes) are added into the μ L of supernatant 25 of above-mentioned absorption
XP reagents, draw piping and druming 5 times up and down, mix DNA is resuspended completely；
(2) it is incubated at room temperature 5 minutes；
(3) it is placed on above magnetic frame, is incubated 3 minutes, until solution clarification, carefully siphons away and discard supernatant, no
Disturb magnetic bead；Note：It should not be abandoned containing amplification library on magnetic bead.
(4) 70% ethanol of 150 μ l Fresh is added, did not had Magnetic bead sample, centrifuge tube both forward and reverse directions movement 5
It is secondary, then it is incubated 2 minutes on magnetic frame, removes supernatant；
(5) repeat the above steps 4, second of washing of progress；
(6) ensure that ethanol drop is all siphoned away from hole, plate be positioned on magnetic frame, air at room temperature is dried 5 minutes,
It is careful not to over-drying.
(7) sample cell is taken away from magnetic frame, 25 μ L TE (PH8.0) buffer solutions is added in every hole and fully infiltrate magnetic
Pearl.Fully vibration is mixed, and liquid is collected into ttom of pipe by quick centrifugation.(it can also select to be drawn on the liquid of more than half with rifle
It is lower to blow and beat at least 5 times to mix)；Note：Supernatant, which contains amplification library, to be abandoned.
Sample cell is placed on magnetic frame 2 minutes.Contain amplified production in supernatant.Take out 20 μ L of supernatant.
The template amount that amplified production obtained by above-mentioned purifying prepares the PCR reactions of library production is 5uL, and remaining component is as follows
Shown in table：
PCR amplification programs set such as following table：
PCR primer is purified by purifying amplified production method respectively, and library production is made.
The detection of above-mentioned library production is specific as follows：
96 parts of samples can once be detected using Ion torrent PGM semiconductors sequenators (Thermofisher companies)
(including yin and yang attribute control).
The system of foregoing 40 parts of whole blood samples and positive plasmid through the present invention detects that only positive plasmid and clinic receives
Tumour radiotherapy has the mutant nucleotide sequence that detects of serious toxicity, and clinic receives tumour radiotherapy without serious toxicity sample
Product further demonstrate the specificity of this method, concrete outcome is as shown in Figure 1 to Figure 3 without mutant nucleotide sequence.
Replica test：Each reaction is separately added into mutational cell line DNA10ng, 1ng and 100pg, is repeated 10 times progress
High-flux sequence detects that 10 times result is consistent, coincidence rate 100%.
Understand that tumour radiotherapy virulent gene library construction reaction of the present invention can just detect tumour radiotherapy simultaneously above
61 hereditary variation sites of virulent gene are treated, the library construction time only needs 3 hours, therefore the present invention is time saving and energy saving, accurately
Property it is high, the quick diagnosis of mutation can be met.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to skill of the invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Claims (5)
1. the construction method of tumour radiotherapy virulent gene mutated library, it is characterised in that covering human gene XRCC1,
TNFa、TXNRD2、ERCC2、TGFB1、VEGF、DNMT1、TP53、XRCC3、LIN28B、MTHFR、PON1、GSTP1、ATM、
NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、IL4、NEIL1、NBN、APC、NFKBIA、DEAD、
It is total on Tpa, RAD51, NFE2L2, GSTA1, MGMT, TDG, GSK3B, FSHR, MYO3B, TGFBR2,5q31, HSPB1 and TXN
Totally 61 kinds of hereditary variation sites, specifically include following steps：
(1) for target gene XRCC1, TNFa, TXNRD2, ERCC2, TGFB1, VEGF, DNMT1, TP53, XRCC3,
LIN28B、MTHFR、PON1、GSTP1、ATM、NOS2、APEX1、MLH1、IL8、CD44、LIG4、IL1A、TNF、CYP2C8、
IL4、NEIL1、NBN、APC、NFKBIA、DEAD、Tpa、RAD51、NFE2L2、GSTA1、MGMT、TDG、GSK3B、FSHR、
MYO3B, TGFBR2,5q31, HSPB1 and TXN design basic amplimer group, the forward primer of the basic amplimer group and anti-
To 5 ' ends of primer provided with 2~5 extra T, and first T close to 3 ' ends of 2~5 T has PNA modifications, simultaneously
The Tm values difference of the basic amplimer group is no more than 1 DEG C；
(2) template, above-mentioned basic amplimer group are placed in the PCR reaction systems containing RingCap-Taq enzymes and expanded
Increase, amplified production is obtained after purification；By amplified production, multipair first asymmetric linking probe, universal primer and above-mentioned RingCap-
Taq enzyme is mixed into performing PCR, and library production is obtained after purification；
(3) library production is constituted into the oncogene variation library for high-flux sequence；
Above-mentioned RingCap-Taq enzymes are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA.
2. the construction method of tumour radiotherapy virulent gene mutated library according to claim 1, it is characterised in that：Institute
Stating basic amplimer group includes FL-XRCC1-1-F, FL-XRCC1-1-R, FL-TNFa-1-F, FL-TNFa-1-R, FL-
TXNRD2-1-F、FL-TXNRD2-1-R、FL-ERCC2-1-F、FL-ERCC2-1-R、FL-TGFB1-1-F、FL-TGFB1-1-R、
FL-VEGF-1-F、FL-VEGF-1-R、FL-DNMT1-1-F、FL-DNMT1-1-R、FL-XRCC1-2-F、FL-XRCC1-2-R、
FL-TP53-1-F、FL-TP53-1-R、FL-XRCC3-1-F、FL-XRCC3-1-R、FL-LIN28B-1-F、FL-LIN28B-1-
R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-1-R、FL-PON1-1-F、FL-PON1-1-
R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、FL-XRCC1-3-F、FL-XRCC3-1-R、
FL-LIN28B-1-F、FL-LIN28B-1-R、FL-XRCC3-2-F、FL-XRCC3-2-R、FL-MTHFR-1-F、FL-MTHFR-
1-R、FL-PON1-1-F、FL-PON1-1-R、FL-GSTP1-1-F、FL-GSTP1-1-R、FL-ATM-1-F、FL-ATM-1-R、
FL-XRCC1-3-F、FL-XRCC1-3-R、FL-NOS2-1-F、FL-NOS2-1-R、FL-TGFB1-2-F、FL-TGFB1-2-R、
FL-APEX1-1-F、FL-APEX1-1-R、FL-VEGF-2-F、FL-VEGF-2-R、FL-XRCC1-4-F、FL-XRCC1-4-R、
FL-MLH1-1-F、FL-MLH1-1-R、FL-IL8-1-F、FL-IL8-1-R、FL-CD44-1-F、FL-CD44-1-R、FL-
LIG4-1-F、FL-LIG4-1-R、FL-IL1A-1-F、FL-IL1A-1-R、FL-TNF-1-F、FL-TNF-1-R、FL-CYP2C8-
1-F、FL-CYP2C8-1-R、FL-IL1A-2-F、FL-IL1A-2-R、FL-IL4-1-F、FL-IL4-1-R、FL-IL4-2-F、
FL-IL4-2-R、FL-NEIL1-1-F、FL-NEIL1-1-R、FL-NBN-1-F、FL-NBN-1-R、FL-APC-1-F、FL-APC-
1-R、FL-NFKBIA-1-F、FL-NFKBIA-1-R、FL-DEAD-1-F、FL-DEAD-1-R、FL-ATM-2-F、FL-ATM-2-
R、FL-IL8-2-F、FL-IL8-2-R、FL-ATM-3-F、FL-ATM-3-R、FL-tPA-1-F、FL-tPA-1-R、FL-TGFB1-
3-F、FL-TGFB1-3-R、FL-RAD51-1-F、FL-RAD51-1-R、FL-NFE2L2-1-F、FL-NFE2L2-1-R、FL-
GSTA1-1-F、FL-GSTA1-1-R、FL-MGMT-1-F、FL-MGMT-1-R、FL-ATM-4-F、FL-ATM-4-R、FL-TDG-
1-F、FL-TDG-1-R、FL-TDG-2-F、FL-TDG-2-R、FL-GSK3B-1-F、FL-GSK3B-1-R、FL-NOS2-2-F、
FL-NOS2-2-R、FL-XRCC1-5-F、FL-XRCC1-5-R、FL-FSHR-1-F、FL-FSHR-1-R、FL-MYO3B-1-F、
FL-MYO3B-1-R、FL-TGFBR2-1-F、FL-TGFBR2-1-R、FL-TGFBR2-2-F、FL-TGFBR2-2-R、FL-5q31-
1-F、FL-5q31-1-R、FL-LIN28B-2-F、FL-LIN28B-2-R、FL-HSPB1-1-F、FL-HSPB1-1-R、FL-NBN-
2-F, FL-NBN-2-R, FL-TXN-1-F and FL-TXN-1-R.
3. the construction method of tumour radiotherapy virulent gene mutated library according to claim 2, it is characterised in that：
XRCC1 gene primer titles：FL-XRCC1-1-F, sequence information TTTTCTGCTGCAGGACACGACA；FL-XRCC1-1-R, sequence
Column information TTTTCGCGCTTGCGCACTTTA；
TNFa gene primer titles：FL-TNFa-1-F, sequence information TTTTCCCTCCCAGTTCTAGTTCTAT；FL-TNFa-1-
R, sequence information TTTTCTTCTGGGCCACTGACTGATT；
TXNRD2 gene primer titles：FL-TXNRD2-1-F, sequence information TTTTCACATTGTCGTAGTCCATCAGA；FL-
TXNRD2-1-R, sequence information TTTTGCAAAGGGTGATGGCATAGG；
ERCC2 gene primer titles：FL-ERCC2-1-F, sequence information TTTTAAGACTCAGGAGTCACCAGGA；FL-
ERCC2-1-R, sequence information TTTTTCTGTTCTCTGCAGGAGGATC；
TGFB1 gene primer titles：FL-TGFB1-1-F, sequence information TTTTGTCAGGCTGGGAAACAAGGTA；FL-
TGFB1-1-R, sequence information TTTTGGTGCTCAGTAAAGGAGAGCAA；
VEGF gene primer titles：FL-VEGF-1-F, sequence information TTTTCCCAAATCACTGTGGATTTTG；FL-VEGF-1-
R, sequence information TTTTCCCAAAAGCAGGTCACTCACT；
DNMT1 gene primer titles：FL-DNMT1-1-F, sequence information TTTTCCCAAACATAATCCCGGACTAT；FL-
DNMT1-1-R, sequence information TTTTTAAGCATGTGCTTTGTTTCCTGT；
XRCC1 gene primer titles：FL-XRCC1-2-F, sequence information TTTTCTGGGACCACCTGTGTTCT；FL-XRCC1-
2-R, sequence information TTTTCCGCATCGTGCGTAAGGA；
TP53 gene primer titles：FL-TP53-1-F, sequence information TTTTGGTTTTCTGGGAAGGGACAGA；FL-TP53-1-
R, sequence information TTTTCGGACGATATTGAACAATGGTTC；
XRCC3 gene primer titles：FL-XRCC3-1-F, sequence information TTTTCGCATCCTGGCTAAAAATACGA；FL-
XRCC3-1-R, sequence information TTTTATTCCGCTGTGAATTTGACAG；
LIN28B gene primer titles：FL-LIN28B-1-F, sequence information TTTTTGAATTAAAACATGTAGCTGCTGAAG；
FL-LIN28B-1-R, sequence information TTTTAAGAAACCTCTTTAACTAGGCATCA；
LIN28B gene primer titles：FL-LIN28B-1-F, sequence information TTTTTGAATTAAAACATGTAGCTGCTGAAG；
FL-LIN28B-1-R, sequence information TTTTAAGAAACCTCTTTAACTAGGCATCA；
XRCC3 gene primer titles：FL-XRCC3-2-F, sequence information TTTTAGTGTCCACTGACGGATAACAGA；FL-
XRCC3-2-R, sequence information TTTTCCTAATCAGCTGTCAAGGGTGAT；
Mthfr gene Primer：FL-MTHFR-1-F, sequence information TTTTCACTCCAGCATCACTCACTTTG；FL-
MTHFR-1-R, sequence information TTTTGGGAGCTGAAGGACTACTACCT；
PON1 gene primer titles：FL-PON1-1-F, sequence information TTTTATACTTGCCATCGGGTGAAATGT；FL-PON1-
1-R, sequence information TTTTGCACTTTTATGGCACAAATGATCAC；
GSTP1 gene primer titles：FL-GSTP1-1-F, sequence information TTTTAGTGACTGTGTGTTGATCAGGC；FL-
GSTP1-1-R, sequence information TTTTAGATGCTCACATAGTTGGTGTAGATG；
ATM gene primer titles：FL-ATM-1-F, sequence information TTTTACTCATTTTTACTCAAACTATTGGG；FL-ATM-
1-R, sequence information TTTTCGAAGACAGCTGGTGAAAAATCC；
XRCC1 gene primer titles：FL-XRCC1-3-F, sequence information TTTTCTACCACACCCTGAAGGATCTTC；FL-
XRCC1-3-R, sequence information TTTTCTAATCTACTCTTTGTCTTCTCCAGT；
NOS2 gene primer titles：FL-NOS2-1-F, sequence information TTTTGGCTAGGAGTAGGACAACGGAA；FL-NOS2-
1-R, sequence information TTTTTGGCCAGGTTTCCAGAAGAAAG；
TGFB1 gene primer titles：FL-TGFB1-2-F, sequence information TTTTGTAACCATCATGGGCCTTGTC；FL-
TGFB1-2-R, sequence information TTTTTGTTCTGAGGACATGGGCAAA；
APEX1 gene primer titles：FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT；FL-
APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC；
APEX1 gene primer titles：FL-APEX1-1-F, sequence information TTTTCTATTGATGCCTAATGCCTGAACT；FL-
APEX1-1-R, sequence information TTTTCGAGTCAAATTCAGCCACAATCAC；
VEGF gene primer titles：FL-VEGF-2-F, sequence information TTTTCACACCATCACCATCGACAGAA；FL-VEGF-
2-R, sequence information TTTTGCAAGAAAAATAAAATGGCGAATC；
XRCC1 gene primer titles：FL-XRCC1-4-F, sequence information TTTTCGAGTCTAGGTCTCAACCCTAC；FL-
XRCC1-4-R, sequence information TTTTCGGGACCTTAGAAGGTGACAGT；
MLH1 gene primer titles：FL-MLH1-1-F, sequence information TTTTCAACCCACAGAGTTGAGAAATTTG；FL-
MLH1-1-R, sequence information TTTTAAGAGCCAAGGAAACGTCTAGATG；
IL8 gene primer titles：FL-IL8-1-F, sequence information TTTTTGTTCTAACACCTGCCACTCTAGT；FL-IL8-1-
R, sequence information TTTTCGGAGTATGACGAAAGTTTTCTTTGA；
CD44 gene primer titles：FL-CD44-1-F, sequence information TTTTGGTCCCTGGGAGGAAATTTGAA；FL-CD44-
1-R, sequence information TTTTGTGTCTCCCAGAAGCATCTGAAAA；
LIG4 gene primer titles：FL-LIG4-1-F, sequence information TTTTAAGAATCTAAAAATTCCCTGAAGTGTCT；FL-
LIG4-1-R, sequence information TTTTTGCTTTACTAGTTAAACGAGAAGATTCAT；
IL1A gene primer titles：FL-IL1A-1-F, sequence information TTTTCAGCCGTGAGGTACTGATCATT；FL-IL1A-
1-R, sequence information TTTTCATTAATCTGCACTTGTGATCATGGTT；
Tnf gene Primer：FL-TNF-1-F, sequence information TTTTGATGTGACCACAGCAATGGGTA；FL-TNF-1-R,
Sequence information TTTTCCCAGTGTGTGGCCATATCTTC；
CYP2C8 gene primer titles：FL-CYP2C8-1-F, sequence information TTTTTCAATTGGGAACAACAGAGTTAACTCC；
FL-CYP2C8-1-R, sequence information TTTTTGGAATTAGTTGGAATTTACATG；
IL1A gene primer titles：FL-IL1A-2-F, sequence information TTTTAGAAGCCAGTGGCTAAGTTTGG；FL-IL1A-
2-R, sequence information TTTTTTCATTTGCTAAGAGTCTGGTGTTCT；
IL4 gene primer titles：FL-IL4-1-F, sequence information TTTTCCCAAGTGACTGACAATCTGGT；FL-IL4-1-R,
Sequence information TTTTCCCATTAATAGGTGTCGATTTGCAGT；FL-IL4-2-F, sequence information
TTTTCCCAAACTAGGCCTCACCTGATA；FL-IL4-2-R, sequence information TTTTGTACAGGTGGCATCTTGGAAACT；
NEIL1 gene primer titles：FL-NEIL1-1-F, sequence information TTTTGGCTTCTCAACTCATGGTCTCT；FL-
NEIL1-1-R, sequence information TTTTGATGGCTTCCCAGGTATTTGGT；
NBN gene primer titles：FL-NBN-1-F, sequence information TTTTATCCTGAAACAAGCATTAAAGAGGGA；FL-NBN-
1-R, sequence information TTTTCGTCCAATTGTAAAGCCAGAATATTTT；
Apc gene Primer：FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT；FL-
APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT；
Apc gene Primer：FL-APC-1-F, sequence information TTTTGCGATGGATATATACGCAGGTAATTTT；FL-
APC-1-R, sequence information TTTTCTTTCTTTGGCTATTTTTGATATGCCT；
NFKBIA gene primer titles：FL-NFKBIA-1-F, sequence information TTTTAGTGTGCAGTGTGGATATAAGTACAC；
FL-NFKBIA-1-R, sequence information TTTTTTTCAGCTGCCCTATGATGACTG；
DEAD gene primer titles：FL-DEAD-1-F, sequence information TTTTTCCTTGCTTCCAGTTTTTCCACT；FL-DEAD-
1-R, sequence information TTTTGGACTTTATCCACTGAAATGTGATA；
ATM gene primer titles：FL-ATM-2-F, sequence information TTTTTGCGTGGCTAACGGAGAAAA；FL-ATM-2-R, sequence
Column information TTTTGGGTCGCACACGACTGAATT；
IL8 gene primer titles：FL-IL8-2-F, sequence information TTTTGCCTACTATAAATAACACTGTGGTA；FL-IL8-
2-R, sequence information TTTTCCCTTGACCTCAGTTAGTTCTTTGTT；
ATM gene primer titles：FL-ATM-3-F, sequence information TTTTAGGGAAATCATAAAGTACGTGAGTCT；FL-ATM-
3-R, sequence information TTTTATGCTTTCTATTTCTCAGCAAAAACA；
TPA gene primer titles：FL-tPA-1-F, sequence information TTTTTAACACTGACAATAACCAAAACCAAG；FL-tPA-
1-R, sequence information TTTTCCCAGGTCTGAGTGATCTCATTG；
TGFB1 gene primer titles：FL-TGFB1-3-F, sequence information TTTTGTAGCCACAGCAGCGGTAGC；FL-TGFB1-
3-R, sequence information TTTTTTCCCTCGAGGCCCTCCTAC；
RAD51 gene primer titles：FL-RAD51-1-F, sequence information TTTTGCAACTCATCTGGGTTGTGC；FL-RAD51-
1-R, sequence information TTTTCTCACACACTCACCTCGGTC；
NFE2L2 gene primer titles：FL-NFE2L2-1-F, sequence information TTTTGGGCTAAAGATTTGGACCCAG；FL-
NFE2L2-1-R, sequence information TTTTGAATGGAGACACGTGGGAGTT；
GSTA1 gene primer titles：FL-GSTA1-1-F, sequence information TTTTATAAGATCAGTACTTACTTTGTTAAA；FL-
GSTA1-1-R, sequence information TTTTGAGTGGCTTTTCCCTAACTTGACT；
Mgmt gene Primer：FL-MGMT-1-F, sequence information TTTTCCCATAGTTCAGTTCTCTGTGTAGG；FL-
MGMT-1-R, sequence information TTTTCTAGCACGAGGTGGGCAGAGT；
ATM gene primer titles：FL-ATM-4-F, sequence information TTTTTCTGTAATCTATAGCAGAGTAAAGCG；FL-ATM-
4-R, sequence information TTTTAAAAACAAAAAGAGCTGACTACCCA；
TDG gene primer titles：FL-TDG-1-F, sequence information TTTTCCCACATTGTTTCCTTTGAACAGA；FL-TDG-1-
R, sequence information TTTTAGTCTATGGCATTCTGAAACAGCAA；FL-TDG-2-F, sequence information
TTTTGTCTGAAGACACTTTTGATGATCAAG；FL-TDG-2-R, sequence information
TTTTCTCAGGACGTAAAGCAGTTTCTCA；
GSK3B gene primer titles：FL-GSK3B-1-F, sequence information TTTTGTTTGCACAACTTCGTGAATCTACTC；FL-
GSK3B-1-R, sequence information TTTTTGACCTTAAGTGCATGGACATTGG；
NOS2 gene primer titles：FL-NOS2-2-F, sequence information TTTTCGCCTCTGACAGATACACTCAG；FL-NOS2-
2-R, sequence information TTTTCAATCTGCAATAACGCAATAGTGCAA；
XRCC1 gene primer titles：FL-XRCC1-5-F, sequence information TTTTCCAGGTCACTTTTGGATTCTGGT；FL-
XRCC1-5-R, sequence information TTTTCCTCATGGTGTGTGTATCAGCA；
FSHR gene primer titles：FL-FSHR-1-F, sequence information TTTTCCGAGAAATCCAGAAATCTAGACTTA；FL-
FSHR-1-R, sequence information TTTTTCTACCCAGTGTATCATTCTGCTTACT；
MYO3B gene primer titles：FL-MYO3B-1-F, sequence information TTTTCATGATCCTTTATACTGCCACCAGAA；FL-
MYO3B-1-R, sequence information TTTTTGGCAAGTTAATGTCCAAGATGGAATT；
TGFBR2 gene primer titles：FL-TGFBR2-1-F, sequence information TTTTTCTGAATGCCCAGAAGACCTCT；FL-
TGFBR2-1-R, sequence information TTTTGGGAAGCATGGAATATGACAAGAGTC；FL-TGFBR2-2-F, sequence information
TTTTCCACATCCATTTGGTGCTTTTAATTGA；FL-TGFBR2-2-R, sequence information
TTTTCCAGTCGATAACTAAGAAGAAGCTACA；
5q31 gene primer titles：FL-5q31-1-F, sequence information TTTTTTGATAACAGCAGAAGATAGAAGAGTT；FL-
5q31-1-R, sequence information TTTTCTGTATTCCCAAGACAAAGCCTCA；
LIN28B gene primer titles：FL-LIN28B-2-F, sequence information TTTTCAATTGTGTGAAGCCAGAGCAT；FL-
LIN28B-2-R, sequence information TTTTACATTCCTACGACATGGAGACAAATG；
HSPB1 gene primer titles：FL-HSPB1-1-F, sequence information TTTTGCAATGCATGACTGTTACCATCATT；FL-
HSPB1-1-R, sequence information TTTTGCAATCTAGTAATCTGGTGTGGTTGTTAG；
NBN gene primer titles：FL-NBN-2-F, sequence information TTTTATTATTTCAGCCAGATGGCAGACT；FL-NBN-2-
R, sequence information TTTTTCTGTAAAATAAGAATAGCAAAGGCAGT；
TXN gene primer titles：FL-TXN-1-F, sequence information TTTTACATACCCAAGCTTCTTAAATGGAAACT；FL-
TXN-1-R, sequence information TTTTCCTTATTTGAAATCCCAGCCTCAGAATT.
4. the construction method of tumour radiotherapy virulent gene mutated library according to claim 1, it is characterised in that：
In step (2), enriched multiple PCR reaction system is：
1 × PCR buffer solutions
5. the structure of the tumour radiotherapy virulent gene mutated library according to any one of Claims 1-4 claim
Construction method, it is characterised in that：The kit of the construction method, including
One DNA is enriched with reaction component, is made up of basic amplimer group；
One RingCap-Taq enzymes, are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA；
One pipe positive quality control product；
With the negative quality-control product of a pipe.
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| WO2020124472A1 (en) * | 2018-12-20 | 2020-06-25 | 深圳华大智造科技有限公司 | Pcr primer, pcr amplification method and use thereof |
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| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| US20130004481A1 (en) * | 2011-01-12 | 2013-01-03 | Boehringer Ingelheim International Gmbh | Anticancer therapy |
| US20130143747A1 (en) * | 2011-12-05 | 2013-06-06 | Myriad Genetics, Incorporated | Methods of detecting cancer |
| CN103370334A (en) * | 2010-12-23 | 2013-10-23 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and method for preparing same |
| CN106498035A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence |
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| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
| CN103370334A (en) * | 2010-12-23 | 2013-10-23 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-I polypeptide or fragment thereof and method for preparing same |
| US20130004481A1 (en) * | 2011-01-12 | 2013-01-03 | Boehringer Ingelheim International Gmbh | Anticancer therapy |
| US20130143747A1 (en) * | 2011-12-05 | 2013-06-06 | Myriad Genetics, Incorporated | Methods of detecting cancer |
| CN106498035A (en) * | 2016-09-30 | 2017-03-15 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence |
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| WO2020124472A1 (en) * | 2018-12-20 | 2020-06-25 | 深圳华大智造科技有限公司 | Pcr primer, pcr amplification method and use thereof |
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