CN108424957A - A kind of cancer of pancreas trace amount DNA enrichment captures method and the application of sequencing - Google Patents
A kind of cancer of pancreas trace amount DNA enrichment captures method and the application of sequencing Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of cancer of pancreas trace amount DNAs to be enriched with the method that capture is sequenced, the method includes the steps：(1) dissociative DNA is extracted from sample；(2) dissociative DNA is built into library；(3) library is enriched with；(4) library by the enrichment captures, and obtains capture dna；(5) capture dna is sequenced, obtains sequencing result；(6) by the sequencing result compared with reference sequences, mutational site is obtained.
Description
Technical field
The invention belongs to cancer gene detection fields, and being enriched with capture more particularly to a kind of cancer of pancreas trace amount DNA surveys
The method of sequence and application.
Background technology
Cancer of pancreas is common one of the malignant tumour of alimentary canal, grade malignancy is high, poor prognosis (Siegel R,
Naishadham D,Jemal A.Cancer statistics,2013.CA:a cancer journal for
clinicians.2013；63(1):11-30.doi:10.3322/caac.21166.).Global pancreas mortality of carcinoma is 4.2%,
The 7th of mortality of malignant tumors is ranked, 5 years survival rates are less than 6%, wherein recurrence and transfer are the masters of Pancreas cancer patients death
Want reason.Currently, clinically generally use iconography, tumor markers, aspiration biopsy method to cancer of pancreas carry out recurrence or
Therapeutic advance is assessed.Due to the characteristic of the weary blood supply of cancer of pancreas, conventional image learns to do the more difficult discovery early stage transfer stoves of Duan Jun and part
Stove is recurred, Radiologic imaging is caused to lag；CA-199 is the finger for cancer of pancreas monitoring after operation of only FDA approvals at present
Mark, but there is also the limitation of sensibility and specificity (Osayi SN, Bloomston M, Schmidt CM, Ellison EC,
Muscarella P.Biomarkers as predictors of recurrence following curative
resection for pancreatic ductal adenocarcinoma:a review.BioMed research
international.2014；2014:468959.doi:10.1155/2014/468959.)；Most of Pancreas cancer patients recurrence
Or the biopsy specimen of metastatic lesion is difficult to obtain, therefore lack at present to the early monitoring index of diagnosis of pancreatic cancer relapse and metastasis and
Technology.
Genetic test based on tissue sample and peripheral blood sample early diagnoses cancer of pancreas, operation minimal residual is examined
Survey, late period medication guide, curative effect monitoring and prognosis evaluation it is with important application prospects (Kenner BJ, Chari ST,
Cleeter DF,Go VL.Early detection of sporadic pancreatic cancer:strategic map
for innovation--a white paper.Pancreas.2015Jul；44(5):686-92.doi:10.1097/
MPA.0000000000000369.；Bettegowda C,Sausen M,Leary RJ.Detection of Circulating
Tumor DNA in Early-and Late-Stage Human Malignancies.Sci Transl Med.2014；6
(224):224ra24doi:10.1126/scitranslmed.3007094.).Since cancer of pancreas enzymatic activity is high, connective tissue is rich
The tissue characteristics such as richness have an impact pancreatic cancer samples DNA extracted amounts, peripheral blood extraction free Tumour DNA amount generally also compared with
It is low, the usage amount of conventional molecular detection cannot be met, such as traditional tissue gene detects --- Sanger is sequenced, and generally requires micro-
The DNA sample of gram-grade.And the method for based on PCR, it is often only capable of judging the mutation in individual gene minority site in one-time detection
Situation, such as KRAS hotspot mutations.It is swum how to efficiently use cast-off cells, aspiration biopsy trace sample, peripheral blood is come from
Genetic test is carried out from the trace amount DNA extracted in DNA sample, is the key that solve the problems, such as cancer of pancreas Molecular Detection.
Therefore, there is a need in the art for a kind of technologies carrying out genetic test using trace amount DNA.
Invention content
The present invention provides one kind in view of the characteristic for being not easy to obtain of pancreatic tissue sample DNA or peripheral blood dissociative DNA
The method of low initial amount DNA enrichments capture sequencing, to overcome the shortcomings of original technology.
Therefore, in a first aspect, the present invention provides a kind of cancer of pancreas trace amount DNA be enriched with capture sequencing method, it is described
Method includes step：
(1) dissociative DNA is extracted from sample；
(2) dissociative DNA is built into library；
(3) library is enriched with；
(4) library by the enrichment is captured using capture probe, obtains capture dna；
(5) capture dna is sequenced, obtains sequencing result；
(6) by the sequencing result compared with reference sequences, mutational site is obtained.
In one embodiment, in step (1), the sample is selected from pancreatic juice cast-off cells, aspiration biopsy sample, saliva
Liquid, pleural effusions and ascites, urine, excrement etc..
In one embodiment, PCR is carried out in step (3) be enriched with the library.The PCR primer, it is preferred to use
High-flux sequence universal primer.
In one embodiment, the gene order of listed gene during capture probe is for table 3 in step (4)
Probe.
In one embodiment, the probe of listed gene mutation during capture probe is for table 4 in step (4).
In one embodiment, the probe of listed gene loci during capture probe is for table 5 in step (4).
In second aspect, the present invention provides a kind of capture chip, the capture chip includes for listed base in table 3
The capture probe of the gene order of cause.
In one embodiment, the probe of listed gene mutation during capture probe is for table 4 in step (4).
In one embodiment, the probe of listed gene loci during capture probe is for table 5 in step (4).
In the third aspect the present invention provides the gene order combination for being used to indicate cancer of pancreas, the assortment of genes includes table
The gene order of listed gene in 3.
In one embodiment, the gene order combination includes gene mutation site listed in table 4.
In one embodiment, the gene order combination includes gene loci listed in table 5.
Relative to other analysis methods, the advantage of method of the invention is as follows：
1) high applicability：Relative to traditional Sanger sequencing approaches, this method only need down to 10ng DNA sample i.e.
Genetic test can be carried out, applicability of the molecular detecting method in pancreatic cancer samples is increased；
2) high-throughput：In conjunction with the experimental method of next-generation sequencing technologies and hybrid capture, can simultaneously to multiple samples into
Row detection, and human pancreatic cancer-associated genes can be disposably detected, more fully abrupt information is provided；
3) high flexibility：Compared to traditional Molecular Detection means, this method may be implemented to single patient, segmentum intercalaris when more
Point sample or multizone sample, are continuously detected so that detection is more flexible；
4) high accuracy：By the sequencing of high depth, the sensibility of detection mutation can be improved, realizes that abundance is 0.1%
The accurate detection of mutation.
Description of the drawings
By the following drawings, the present invention will be described：
Fig. 1 cancer of pancreas trace amount DNAs enrichment capture sequencing schematic diagram.
Specific implementation mode
The purpose of the present invention is to provide a kind of efficiently gene being carried out using Pancreas cancer patients sample (tissue or peripheral blood)
The method of detection, this method provides the preparation methods of trace amount DNA (nanogram level) sample, and provide the library construction side of optimization
The enrichment chip of method and autonomous Design carries out efficient capture to DNA library, further uses the method for high-flux sequence to more bases
Because region is detected (Fig. 1), pancreas oncogene mutation state can efficiently, be accurately, flexibly assessed, and may further be right
Cancer of pancreas early diagnosis, molecular targeted medication and recurrence monitoring are assessed.
In the present invention, Gene Name is all made of NCBI-Gene (https://www.ncbi.nlm.nih.gov) it is inner
Official names (Official Symbol).The reference sequences of chromosome sequence are GRCh37/hg19 (http://
Hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/), the chromosome involved in the present invention is compiled
Number and position be also based on reference sequences be GRCh37/hg19.
In the present invention, the method for detection gene mutation site is not related to the diagnosis of disease merely, but dashes forward with certain genes
The method for becoming the detection gene mutation that site is combined can be used for disease detection.Therefore, the method for the present invention includes non-diagnostic
Method.
1. capture probe designs：
The abrupt information of 598 Pancreas cancer patients is carried out confluence analysis by the experience accumulation in being worked according to inventor, into
Row capture chip design, the high frequency exon region of the exon region of 43 High frequency genes and 5 hot spot genes is designed into
Chip, such as following table.
Table 1：Cancer of pancreas common mutations list of genes
2. trace amount DNA extracting method
2.1 pancreatic juice cast-off cells DNA extraction methods
Using ERCP art indwelling nose catheter collections pancreatic juice 24 hours, receiving flask surrounding placed ice cube.Pancreatic juice amount is not less than
It is spare to be stored in -80 DEG C of refrigerators by 5ml.
2.1.1 pancreatic juice cast-off cells separation process
1ml pancreatic juice is moved into 1.5ml centrifuge tubes, 1000g centrifuges 5min, abandons supernatant.The sterile lifes of 1ml are added in precipitation
Reason brine mixes well, and 1000g centrifuges 5min, discards supernatant, it is spare to be stored in -80 DEG C of refrigerators.
2.1.2 pancreatic juice cast-off cells DNA is extracted
Pancreatic juice cast-off cells are completed using QIAamp Micro DNA Kit (QIAGEN) DNA extraction kit.
2.2 dissociative DNA in blood extracting methods
Using EDTA anti-freezing blood sampling tubes, anticoagulant heparin must not be used；Blood sampling volume is not less than 8ml, runs up and down immediately after blood sampling
Mixing, 2h is interior to complete blood plasma separation.
2.2.1 blood plasma separation process：
(1) by heparin tube, 1600g centrifuges 10min under the conditions of 4 DEG C, and upper plasma is dispensed into multiple 1.5mL after centrifugation
Or in the centrifuge tube of 2.0mL, pay attention to that intermediate leukocytic cream cannot be drawn onto during drawing blood plasma；Lower layer's lymphocyte and blood
Sample is preserved cell as a contrast.
(2) plasma sample dispensed is subjected to secondary separation, 16000g centrifuges 10min under the conditions of 4 DEG C, and supernatant is turned
It moves on in the centrifuge tube of new 1.5mL or 2.0mL, discards residual white；Blood plasma and control sample label after detaching twice
It is clear, it is spare to be stored in -80 DEG C of refrigerators.
2.2.2 plasma DNA extracts
Plasma DNA is extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN) dissociative DNA
Kit is completed.
2.3 aspiration biopsy trace sample DNA extractions
Aspiration biopsy sample is obtained using EUS-FNA or liver puncture, sample weight must not be less than 20mg, in liquid nitrogen quickly
It freezes spare.
Aspiration biopsy trace sample DNA is completed using QIAamp Micro DNA Kit (QIAGEN) DNA extraction kit.
The present invention can use the sample type of other extractable dissociative DNAs, such as saliva, pleural effusions and ascites, urine, excrement
Deng；The peripheral blood acquisition that other heparin tubes carry out；Other methods collect pancreatic juice and biopsy sample；The DNA that other kits carry out
Extraction.
3. library construction：
Dissociative DNA after extraction builds library specification according to KAPA LTP Library Preparation Kit later, into
3 step enzymatic reaction of row.
It repairs 3.1 ends
Later, 120 μ L of Agencourt AMPure XP reagents are added, carry out magnetic beads for purifying, 42 μ L of last back dissolving
ddH2O, band magnetic bead carry out next step reaction.
3.2 plus A reactions
90 μ L of PEG/NaCl SPRI solution are added later, are sufficiently mixed, carry out magnetic beads for purifying, last back dissolving (35- connectors)
μL ddH2O, band magnetic bead carry out next step reaction.
3.3 connectors (Adapter) connect：
It is separately added into 50 μ L of PEG/NaCl SPRI solution later 2 times, carries out 2 magnetic beads for purifying, 25 μ L of last back dissolving
ddH2O。
The enrichment of 3.4 linking libraries
Enrichment PCR is carried out to library using the enrichment method of optimization, amplification enzyme selects Phusion Hot Start
Polymerase, and DMSO is added to eliminate influence of the DNA secondary structures to amplification, specific reaction system is as follows.It is enriched in PCR
Use the primer with joint sequence complementation.The primer of the PCR is high-flux sequence universal primer.The present inventor is to reaction system
The optimization of progress, including polymerase be selected as Phusion rather than KAPA is serial, in addition also have be added DMSO, increase trace
Measure DNA degree of detection.
Using fluorescence quantitative PCR instrument (ABI 7500), condition setting is such as
Agencourt AMPure XP reagents 50uL is added later and carries out magnetic beads for purifying.
It is sequenced with upper machine 4. probe is captured
4.1 high-throughput captures sequencings are to carry out probe covering per~100bp according to reference sequences according to list of genes,
Designed successively from front to back every the probe that 10bp designs a 100bp in the sections certain gene such as 1~3kb.After amplification
After library Quality Control qualification and using inventor design capture probe, with reference to chip manufacturer (Roche) provide specification into
Row hybrid capture.Finally 21 μ L ddH of elution back dissolving2O band hybridization elution magnetic beads.
The amplification of 4.2 hybrid capture products
FellowCell Primer 1 refer to the sequence with the joint sequence complementation for capturing sequence one end institute band；
FellowCell Primer 2 refer to the sequence with the joint sequence complementation for capturing sequence one end institute band.Primers F ellowCell
Primer 1 and FellowCell Primer 2 meets the design principle of the sequence as primer.
4.3 first remove previous step magnetic bead, then rejoin 50 μ L of Agencourt AMPure XP reagents, carry out magnetic bead
Purifying, 25 μ L ddH of last back dissolving2O carries out QC and upper machine.
4.4 carry out machine sequencing using Illumina HiSeq3000PE151 programs, and sequencing experimental implementation is according to manufacturer
The operational manual (announcing cBot referring to Illumina/Solexa officials) of offer carries out upper machine sequencing procedures.
In the present invention, other kits can also be used to carry out library construction.
5. data processing and mutation detection
5.1 times machine data are filtered, and reject low-quality read；
5.2 times machine data are compared using BWA softwares to reference gene group；
5.3 compare the label that bam files carry out repetitive sequence using Picard；
5.4 the bam files after deduplication carry out the place of IndelRealigner and BaseRecalibrator using GATK
Reason；
5.5 mutation detections：Somatic SNV variation detections are carried out with Mutect softwares；Somatic is carried out with GATK is soft
InDel variation detections；It is detected with CONTRA softwares CNV；Used screening parameter is：Normal aberration rate≤2%, variation
Number >=5 reads, p value≤0.05 somatic；
5.6 variation annotations：Detection variation is annotated using ANNOVAR softwares, notes content includes base mutation, ammonia
Base acid mutation, functional mutant etc..
In the present invention, other existing softwares or the personal bam file generateds for writing script progress can also be used, dashed forward
Become detection, mutation annotation.
The source of related software：
1.BWA：http://bio-bwa.sourceforge.net/
2.Picard：https://broadinstitute.github.io/picard/
3.GATK:https://www.broadinstitute.org/gatk/
4.Mutect:http://www.broadinstitute.org/cancer/cga/mutect
5.CONTRA:http://contra-cnv.sourceforge.net/
6.ANNOVAR:http://annovar.openbioinformatics.org/en/latest/
7.SAMtools:http://samtools.sourceforge.net/
Embodiment
The pancreatic juice cast-off cells DNA sample detection for the Pancreas cancer patients for thering is surgical tissue to sample below by specific 50
Acetonideexample, the present invention will be described, it should be noted that the embodiment is merely to illustrate that purpose, and cannot be to appoint
Where formula is construed to limitation of the present invention.50 all volunteers of Pancreas cancer patients, age 25-60 Sui, gender with
Machine, no other diseases.As a contrast, have chosen 100 similar normal controls of age-sex blood sample carried out it is parallel
Detection.
1. the DNA extractions of pancreatic juice cast-off cells：
Using ERCP art indwelling nose catheter collections pancreatic juice 24 hours, receiving flask surrounding placed ice cube.Pancreatic juice amount is not less than
It is spare to be stored in -80 DEG C of refrigerators by 5ml.
1.1 pancreatic juice cast-off cells separation process
Pancreatic juice is dispensed into multiple 1.5ml centrifuge tubes, 1000g centrifuges 5min, abandons supernatant.In precipitation be added 1ml without
Bacterium physiological saline mixes well, and 1000g centrifuges 5min, discards supernatant, it is spare to be stored in -80 DEG C of refrigerators.
1.2 pancreatic juice cast-off cells DNA extractions
Pancreatic juice cast-off cells are completed using QIAamp Micro DNA Kit (QIAGEN) DNA extraction kit.
2. the preparation in sample library
DNA after extraction builds library specification according to KAPA LTP Library Preparation Kit later, carries out 3
Walk enzymatic reaction.
It repairs 2.1 ends
Later, 120 μ L of Agencourt AMPure XP reagent are added, carry out magnetic beads for purifying, 42 μ L of last back dissolving
ddH2O, band magnetic bead carry out next step reaction.
2.2 plus A reactions：
90 μ L of PEG/NaCl SPRI solution are added later, are sufficiently mixed, carry out magnetic beads for purifying, last back dissolving (35- connectors)
μL ddH2O, band magnetic bead carry out next step reaction.
2.3 connectors (Adapter) connect：
It is separately added into 50 μ L of PEG/NaCl SPRI solution later 2 times, carries out 2 magnetic beads for purifying, 25 μ L of last back dissolving
ddH2O。
The enrichment of 2.4 linking libraries
Enrichment PCR is carried out to library using the enrichment method of optimization, amplification enzyme selects Phusion Hot Start
Polymerase, and DMSO is added to eliminate influence of the DNA secondary structures to amplification, specific reaction system is as follows.It is enriched in PCR
Use high-flux sequence universal primer.
The optimization that the present inventor carries out reaction system, including polymerase be selected as Phusion rather than KAPA is serial,
In addition also have and DMSO is added, increase Trace DNA analysis degree.
Using fluorescence quantitative PCR instrument (ABI 7500), condition setting is such as
Agencourt AMPure XP reagents 50uL is added later and carries out magnetic beads for purifying.
It is sequenced with upper machine 3. probe is captured
3.1 high-throughput captures sequencings are to carry out probe covering per~100bp according to reference sequences according to list of genes,
Designed successively from front to back every the probe that 10bp designs a 100bp in the sections certain gene such as 1~3kb.After amplification
After library Quality Control qualification and using inventor design capture probe, with reference to chip manufacturer (Roche) provide specification into
Row hybrid capture.Finally 21 μ L ddH of elution back dissolving2O band hybridization elution magnetic beads.
The amplification of 3.2 hybrid capture products
FellowCell Primer 1 refer to the sequence with the joint sequence complementation for capturing sequence one end institute band；
FellowCell Primer 2 refer to the sequence with the joint sequence complementation for capturing sequence one end institute band.Primers F ellowCell
Primer 1 and FellowCell Primer 2 meets the design principle of the sequence as primer.
3.3 first remove previous step magnetic bead, then rejoin 50 μ L of Agencourt AMPure XP reagents, carry out magnetic bead
Purifying, 25 μ L ddH of last back dissolving2O carries out QC and upper machine.
3.4 carry out machine sequencing using Illumina HiSeq3000PE151 programs, and sequencing experimental implementation is according to manufacturer
The operational manual (announcing cBot referring to Illumina/Solexa officials) of offer carries out upper machine sequencing procedures.
4. positive and negative double-strand error correction low-frequency information analysis：
4.1 times machine data are filtered, and reject low-quality read；
4.2 times machine data are compared using BWA softwares to reference gene group；
4.3 compare the label that bam files carry out repetitive sequence using Picard；
Bam files after 4.4 deduplications carry out the place of IndelRealigner and BaseRecalibrator using GATK
Reason；
4.5 mutation detections：Somatic SNV variation detections are carried out with Mutect softwares；Somatic is carried out with GATK is soft
InDel variation detections；It is detected with CONTRA softwares CNV；Used screening parameter is：Normal aberration rate≤2%, variation
Number >=5 reads, p value≤0.05 somatic；
4.6 variation annotations：Detection variation is annotated using ANNOVAR softwares, notes content includes base mutation, ammonia
Base acid mutation, functional mutant etc..
5. testing result, mutation verification and analysis
50 patients detect that the mutation of 90 genes, the appearance of testing result such as following table provide altogether：
Table 2：The mutation of patient detects result
For all patients, is sampled using its surgical tissue and carry out DNA extractions, and amplification survey is carried out to the gene in table 1
Sequence verifies the result of the method for aforementioned present invention.The result shows that the experiment that method of the invention is obtained with conventional method
As a result it is consistent completely.These mutation are distributed in following gene and exon.
Table 3：Cancer of pancreas common mutations list of genes
| KRAS | CTNNB1 | MLL2 |
| TP53 | MLL3 | PBRM1 |
| MLL | SMAD4 | SPEN |
| CDKN2A | ATRX | CIC |
| DAXX | POLE | NOTCH2 |
| NCOR1 | MED12 | KEAP1 |
| ARID1A | PIK3CA | CBL |
| TERT | TSC1 |
Table 4：It is mutated list
Table 5：It is mutated the position in chromosome
The blood sample of 100 normal controls has carried out the mutation that related locus is not detected in Parallel testing, illustrates that these are prominent
Change is sufficient to differentiating pancreatic cancer and normal individual.
According to the above results, inventor devises a kind of capture chip of simplification, and the capture chip includes in table 5
The probe of listed gene loci.Specific design method, which is sketched, is：Based on the sequence of listed gene loci in table 5, according to ginseng
It examines sequence and carries out probe covering per~100bp, every the probe that 10bp designs a 100bp, design successively from front to back.It repeats
Above-described embodiment, and in step 3.1, it will be after Quality Control qualification in library after amplification and using the capture probe of these simplification, ginseng
Hybrid capture is carried out according to the specification that chip manufacturer (Roche) provides and carries out follow-up sequencing steps, has been obtained and above-mentioned implementation
The identical result of example.Therefore, using simplified sequence this hair can be realized in the case where reducing synthesis, experiment, sequencing cost
Bright purpose.
It is regardless of and is limited to any theory, the present inventor is by the optimization that is carried out to reaction system and selection to suitable gene order
The detection of combination realizes the detection of trace amount DNA (nanogram level) sample of cancer of pancreas.Reaction system is optimized including polymerization
Enzyme be selected as Phusion rather than KAPA is serial, in addition also have be added DMSO, increase Trace DNA analysis degree.Parallel progress
Check experiment, do not reach the detection result in the embodiment of the present invention.
Claims (10)
1. a kind of method of cancer of pancreas trace amount DNA enrichment capture sequencing, the method includes the steps：
(1) dissociative DNA is extracted from sample；
(2) dissociative DNA is built into library；
(3) library is enriched with；
(4) library by the enrichment is captured using capture probe, obtains capture dna；
(5) capture dna is sequenced, obtains sequencing result；
(6) by the sequencing result compared with reference sequences, mutational site is obtained.
2. method of claim 1, in step (1), the sample is selected from pancreatic juice cast-off cells, aspiration biopsy sample, saliva, chest
Seroperitoneum, urine, excrement etc..
3. the method for claims 1 or 2 carries out the PCR enrichments library in step (3).
4. the method for claims 1 or 2, the gene order of listed gene during capture probe is for table 3 in step (4)
Probe.
5. the method for claims 1 or 2, the spy of listed gene mutation during capture probe is for table 4 in step (4)
Needle.
6. the method for claims 1 or 2, the spy of listed genomic locus during capture probe is for table 5 in step (4)
Needle.
7. a kind of capture chip, the capture chip includes the capture probe for the gene order of listed gene in table 3.
8. the capture chip of claim 7, the capture probe is the probe for gene mutation listed in table 4.
9. the capture chip of claim 7 or 8, the capture probe is the probe for genomic locus listed in table 5.
10. being used to indicate the gene order combination of cancer of pancreas, the assortment of genes includes the gene sequence of listed gene in table 3
It arranges, listed gene mutation site preferably in table 4, listed gene loci in more preferable table 5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104611410A (en) * | 2013-11-04 | 2015-05-13 | 北京贝瑞和康生物技术有限公司 | Noninvasive cancer detection method and its kit |
| CN106065414A (en) * | 2016-06-15 | 2016-11-02 | 浙江大学 | Noninvasive cancer of pancreas polygenes detection method and kit based on blood plasma cfDNA detection technique |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611410A (en) * | 2013-11-04 | 2015-05-13 | 北京贝瑞和康生物技术有限公司 | Noninvasive cancer detection method and its kit |
| CN106065414A (en) * | 2016-06-15 | 2016-11-02 | 浙江大学 | Noninvasive cancer of pancreas polygenes detection method and kit based on blood plasma cfDNA detection technique |
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