CN106591288A - Second-generation sequencing library building technology based on probe capturing - Google Patents
Second-generation sequencing library building technology based on probe capturing Download PDFInfo
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Abstract
The invention discloses a second-generation sequencing library building technology based on probe capturing. According to the technology, capturing probes in alternative covering are designed aiming at gene segments or DNA sequences of target detection, the synthesized labeling probes are mixed to form a probe pool, the probe pool is hybridized with sample DNA, by capturing DNA fragments combined with the labeling probes and enriching the DNA fragments, a specific enrichment product of a target segment is obtained, and thus the purpose of sequencing the target segment is obtained. With the adoption of the technology, the sequencing accuracy is improved, the cost of the whole flow is reduced, and the uniform and stable covering degree is achieved.
Description
Technical field
The present invention relates to biological technical field, the two generations sequencing database technology of more particularly to a kind of probe capture
Background technology
The sequencing of two generations has particularly important application, its core due to the sequencing ability of its superelevation in scientific research and clinic
Thought is that (Sequencing by Synthesis) is sequenced in synthesis, is determined by catching the mark of the new end for synthesizing
The sequence of DNA, existing technology platform mainly have Roche/454 FLX, Illumina/Solexa Genome Analyzer and
Applied Biosystems SOLID system。
Polymerase chain reaction (i.e. PCR) is a kind of technology for being widely used in molecular genetic and diagnosis, can be analyzed any
Short DNA sequence dna, even if comprising only low-level DNA in sample.Round pcr can be used for the DNA or RNA of Amplification Analysis and select
Fragment.
Probe capture is built storehouse method and refers to that prepare DNA the or RNA oligomer with chemical labeling carries out spy with target DNA fragments
Specific hydrogen combines and is captured a kind of banking process of collection.Target sequence capture be according to target gene group sequence, if
The probe of complete complementary therewith is counted, these probes are fixed on some holders, then interrupt genomic DNA, plus top connection
For hybridizing with probe after sequencing, the DNA on non-hybridized is eluted, reclaim target DNA fragments, can directly build storehouse carries out DNA surveys
Sequence.Magnetic laws on arrests is the specific bond and base complementrity principle based on biotin and Avidin, by the special widow of biotin labeling
Nucleotide probe is bound on the magnetic bead of coating Avidin, in conjunction with the target nucleotide in the magnetic bead and liquid phase for having specific probe
Hybridization, is then collected using magnetic frame, the washing of Jing low salt buffers, removes the impurity such as isolating protein, non-target nucleotide, is finally given
The specific nucleic acid molecules of purifying.The advantage of the solution hybridization adopted in magnetic laws on arrests is that hybridization efficiency is higher, it is easy to operated, when
Between it is short, be easy to automation mechanized operation.
The reagent kit product such as the RNA capture probes of Agilent in the market and the DNA capture probes of Roche is main logical
The target zone crossed in the oligomer molecular probe of DNA or RNA and the sample DNA of fragmentation carries out the combination of specificity, from
And target fragment is obtained, and the technology for it being enriched with and being sequenced.These products are generally more costly, and on target zone target rate it is low and
Technological process is complicated.
The content of the invention
In view of problem above, the invention provides a kind of two generations sequencing database technology of probe capture, the technology it is basic
Function is that whole database technology is mainly made up of the capture probe reagent of DNA fragmentation plus linker reagents and target fragment, pin
Constant gene segment C or DNA sequence dna to target detection designs the capture probe of alternate covering, and the label probe of synthesis is mixed into
Probe cell, is hybridized with sample DNA, and by capturing the DNA fragmentation combined with label probe, and being enriched with a mesh is obtained
The specific enrichment product of mark section, so as to reach the purpose of target zone sequencing.The technology can improve the precision of sequencing,
The background noise of non-specific fragment is reduced, the data volume of two generations sequencing, and then the cost of reduction whole flow process is reduced, can be reached
The coverage of stable homogeneous.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides experimental program step is as follows：
Step 1, prehybridization experiment, improves the capture rate of capture probe；
Step 2, hybrid capture is hybridized with sample DNA, by capturing the DNA fragmentation combined with label probe；
Step 3, recovery product amplification, is enriched with, and obtains the specific enrichment product of a target zone.
In above scheme, preferably, carrying out prehybridization by pre hyb, PCR programmings are as follows：95 DEG C, 5min；
65 DEG C, 5min；65 DEG C, Hold lockings.
In above scheme, preferably, during hybrid capture, 25uL reaction systems are formulated as follows：Blocking
Buffer 2.75uL, Library 4.25uL, 2 times of hyb buffer 13uL, Probe 5uL.
In above scheme, preferably, in recovery product amplification procedure, 50uL reaction systems are formulated as follows：1mmol/L
DNTP 10uL, 5 times of Phusion buffer 10uL, 10umol/L P5 primer2.5uL, 10umol/L P7 primer
2.5uL, Phusion hot start polymerase 0.5uL, DNA 8uL, NFW 16.5uL.PCR instrument setting program is such as
Under：98 DEG C, 2min；98 DEG C, 2min；98 DEG C, 58 DEG C of 15s, 72 DEG C of 30s, 15s carries out 18 circulations；72 DEG C, 5min；4 DEG C,
Hold is locked, and carries out amplified library.Wherein：
P7 primer：5’AATGATACGGCGACCACCGAGATCT 3’
P5 primer：5’ATCTCGTATGCCGTCTTCTGCTTG 3’
The advantages of the present invention are：A kind of two generations sequencing database technology of probe capture is provided.Using spy
The probe of the opposite sex improves the precision of sequencing capturing target fragment；Capture and the target fragment of enrichment specificity, it is possible to decrease non-
The background noise of specific fragment, so as to reduce the data volume of two generations sequencing, and then reduces the cost of whole flow process；To all targets
Fragment carries out alternate covering design probe to reach the coverage of stable homogeneous.
Specific embodiment
With reference to embodiment, the specific embodiment of the present invention is further described.Following examples are only used for more
Plus technical scheme is clearly demonstrated, and can not be limited the scope of the invention with this.
The prehybridization of embodiment 1.
A) 2 × hyb buffer are placed in into 65 DEG C of baking oven preheatings.Take out blocking buffer in -20 DEG C of refrigerators to be placed in
It is standby on ice.
B) prehybridization is carried out by pre hyb.PCR programmings are as follows：95 DEG C, 5min；65 DEG C, 5min；65 DEG C, Hold
Locking.
C) during pre hyb, probe is taken out from -80 DEG C of refrigerators, takes 1 new EP pipe, configure hybridization system
6uL, matches somebody with somebody according to the 110% of theoretical value, adds 5uL probes, concussion to mix, be centrifuged in short-term, be labeled as hyb-mix and be put into 65 DEG C of bakings
Case preheats 2min.
The hybrid capture of embodiment 2.
After prehybridization terminates, sample is taken out, often pipe rapidly joins 13uL2 × hyb buffer and 6uL hyb- of preheating
Mix, mixes, and is placed in after of short duration centrifugation in PCR instrument, and overnight, whole reaction system configuration is as follows for 65 DEG C of constant-temperature incubations：
The recovery product of embodiment 3. is expanded
A) reagent being first configured in addition to DNA, matches somebody with somebody according to the 110% of theoretical value, and concussion is mixed, and is centrifuged in short-term, dispenses
To in 8 unions, per hole 42uL is added, shift the sample of 8uL from above centrifuge tube afterwards in every hole, closed the lid.Concussion is mixed
It is even, it is centrifuged in short-term.Prepare reaction system as follows：
Wherein：
P7 primer：5’AATGATACGGCGACCACCGAGATCT 3’
P5 primer：5’ATCTCGTATGCCGTCTTCTGCTTG 3’
B) PCR instrument is opened, setting program is as follows：98 DEG C, 2min；98 DEG C, 2min；98 DEG C, 58 DEG C of 15s, 72 DEG C of 30s,
15s carries out 18 circulations；72 DEG C, 5min；4 DEG C, Hold lockings carry out amplified library.
C) after PCR terminates, sample is taken out, of short duration centrifugation, the magnetic bead of 75uL is added in each hole, cover new lid.Shake
Mixing is swung, is centrifuged in short-term.5min is incubated under room temperature.
D) 8 unions are put into magnetic frame, waits 5min, after solution clarification, supernatant is abandoned in suction；The second of 180uL 80% is added per hole
Alcohol, stands 30s, and suction is abandoned supernatant, come again.
E) 3-5min is dried, water droplet on tube wall is broken up in order to evaporate with pipette tips.Observation magnetic bead surfaces are no longer moist anti-
Light, mist formation planar can leave magnetic frame, add 25uL NFW, piping and druming to mix resuspended magnetic bead per hole, be incubated at room temperature 1min.
F) orifice plate puts again magnetic frame, waits 2min, and after solution clarification, transfer supernatant is in new orifice plate.Repeat one
It is secondary.
G) analyze experimental result by bioinformatics software to obtain, the coverage of sequencing reaches 50 up to 100%, 91% × depth
Degree, 76% reach 100 × depth.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Mei Diweikang bio tech ltd
<120>The two generations sequencing database technology of probe capture
<130> 123
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>It is artificial synthesized
<400> 1
aatgatacgg cgaccaccga gatct 25
<210> 2
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 2
atctcgtatg ccgtcttctg cttg 24
Claims (5)
1. the two generations sequencing database technology that a kind of probe is captured, it is characterised in that comprise the steps：
Step 1, prehybridization experiment, improves the capture rate of capture probe；
Step 2, hybrid capture is hybridized with sample DNA, by capturing the DNA fragmentation combined with label probe；
Step 3, recovery product amplification, is enriched with, and obtains the specific enrichment product of a target zone.
2. the two generations sequencing database technology that probe according to claim 1 is captured, it is characterised in that the prehybridization process
In PCR programmings it is as follows：95 DEG C, 5min；65 DEG C, 5min；65 DEG C, Hold lockings.
3. the two generations sequencing database technology that probe according to claim 1 is captured, it is characterised in that the hybrid capture mistake
Cheng Zhong, 25uL reaction system is formulated as follows：2.75uL, Library4.25uL, 2 times of hyb buffer of Blocking buffer
13uL, Probe 5uL.
4. the two generations sequencing database technology that probe according to claim 1 is captured, it is characterised in that described, 50uL reactions
System is formulated as follows：1mmol/L dNTP 10uL, 5 times of Phusion buffer 10uL, 10umol/L P5primer
2.5uL, 10umol/L P7primer 2.5uL, Phusion hot start polymerase 0.5uL, DNA 8uL, NFW
16.5uL。
5. the two generations sequencing database technology that probe according to claim 1 is captured, it is characterised in that the recovery product expands
PCR instrument setting program is as follows in increasing process：98 DEG C, 2min；98 DEG C, 2min；98 DEG C, 15s；58 DEG C, 30s；72 DEG C, 15s is carried out
18 circulations；72 DEG C, 5min；4 DEG C, Hold lockings carry out amplified library.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486101A (en) * | 2018-04-03 | 2018-09-04 | 艾吉泰康生物科技(北京)有限公司 | A kind of method that fast Acquisition builds library |
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| CN102586229A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for preparing capture probe and application of capture probe |
| CN104278022A (en) * | 2014-09-12 | 2015-01-14 | 中国人民解放军第四军医大学 | Preparation method and application of DNA capturing probe |
| CN104789687A (en) * | 2015-05-08 | 2015-07-22 | 上海允英医疗科技有限公司 | Probe preparation method for multi-gene capture sequencing |
| CN105648067A (en) * | 2016-02-19 | 2016-06-08 | 北京乐普基因科技股份有限公司 | Kit for construction of cardiovascular and cerebrovascular disease library, method and application of kit |
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|---|---|---|---|---|
| CN102586229A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for preparing capture probe and application of capture probe |
| CN104278022A (en) * | 2014-09-12 | 2015-01-14 | 中国人民解放军第四军医大学 | Preparation method and application of DNA capturing probe |
| CN104789687A (en) * | 2015-05-08 | 2015-07-22 | 上海允英医疗科技有限公司 | Probe preparation method for multi-gene capture sequencing |
| CN105648067A (en) * | 2016-02-19 | 2016-06-08 | 北京乐普基因科技股份有限公司 | Kit for construction of cardiovascular and cerebrovascular disease library, method and application of kit |
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Cited By (1)
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|---|---|---|---|---|
| CN108486101A (en) * | 2018-04-03 | 2018-09-04 | 艾吉泰康生物科技(北京)有限公司 | A kind of method that fast Acquisition builds library |
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Application publication date: 20170426 |