CN109777858A - The probe and method of hybrid capture are carried out to Duplication region - Google Patents
The probe and method of hybrid capture are carried out to Duplication region Download PDFInfo
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- CN109777858A CN109777858A CN201811564939.5A CN201811564939A CN109777858A CN 109777858 A CN109777858 A CN 109777858A CN 201811564939 A CN201811564939 A CN 201811564939A CN 109777858 A CN109777858 A CN 109777858A
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Abstract
The invention discloses probes and method that a kind of pair of Duplication region carries out hybrid capture.Wherein, which is the primed probe with detection label using specific locations in target gene repeat region side conservative region or repeat region as 3 ' ends.It applies the technical scheme of the present invention, the principle extended using primer amplification, base pair complementarity is greater than in hybrid capture to the specific requirements of sequence to the specific requirements of sequence especially 3 ' end sequences using primer extend, opposite specific locations require highest 3 ' end as primer specificity using in target gene repeat region side conservative region or repeat region, designing has the primed probe of detection label as capture probe, in conjunction with Traditional liquid phase hybrid capture system, the capture rate in Duplication region is specifically improved.
Description
Technical field
The present invention relates to field of biotechnology, carry out hybrid capture in particular to a kind of pair of Duplication region
Probe and method.
Background technique
Solution hybridization capture technique is developed according to base complementrity principle, and target DNA fragments in the solution are passed through
The probe direct cross for having had biotin labeling, is then anchored target DNA fragments by the reaction of biotin-labeled pentylamine
On the magnetic bead with Avidin.Magnetic bead is formed by magnetic particle and high-purity Streptavidin covalent bond, can be used for capturing life
The substrate of object element label, the interaction of biotin-Streptavidin is very strong, and non-specific binding rate is very low, so that captured
Substrate can meet subsequent experimental requirement.Non-targeted DNA is washed away by cleaning magnetic bead, then destination region is obtained by elution magnetic bead
DNA。
There is a large amount of repetitive sequences in human genome, and repetitive sequence often has very high copy number, in gene
Competitive hybridization can occur with target fragment in group hybrid experiment, real aim sequence hybridization efficiency is caused to reduce.People Cot-
1DNA is placenta dna, and length is mainly 50 to 300bp, is rich in duplicate DNA sequence dna, such as Alu and Kpn family member.Therefore
During conventional hybridization, repetitive sequence in block gene group is used for often through Human Cot1DNA is added as sealer
To improve capture rate.But repetitive sequence is equally existed in the region of the important cancer related gene in part, although these regions
It is the destination region it is desirable that being captured, but in traditional hybrid capture technology, in order to reach the excellent of overall performance
Change, generally require " to sacrifice " repetitive sequence in these important areas, the design of probe is not carried out for these regions.Namely
It says, existing hybrid capture technology can not be designed probe to the high repetitive sequence in target gene region or even design
Probe, capture rate be not also high.
Summary of the invention
The present invention is intended to provide a kind of pair of Duplication region carries out the probe and method of hybrid capture, it is existing miscellaneous to solve
Hand over the technical issues of can not carrying out probe design and effectively capture in catching method to high repeat region.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of pair of Duplication region progress is miscellaneous
Hand over the probe of capture.The probe is using specific locations in target gene repeat region side conservative region or repeat region as 3 '
The primed probe with detection label of end.
Further, 5 ' ends of primed probe have Illumina P5 joint sequence.
Further, the biotin modification that detection is added labeled as the 5 ' ends in primed probe.
Further, the length of primed probe is 63bp~73bp, preferably 68bp.
Further, primed probe can match special in target gene repeat region side conservative region or repeat region
The length that dystopy is set is 5bp~20bp, preferably 10bp.
Further, target gene is ROS1 gene, and Duplication region is No. 31 and includes subregion.
Further, the primed probe for including subregion progress hybrid capture to ROS1 gene 31 is as shown in table 1 below:
Table 1
Further, specificity capture Duplication region is used alone in primed probe, or with conventional polygenic capture
Probe cell is used in mixed way.
According to another aspect of the present invention, the method that a kind of pair of Duplication region carries out hybrid capture is provided.The party
Method carries out hybrid capture to Duplication region using any of the above-described kind of probe.
Further, hybrid capture is carried out using solution hybridization capture system, is carrying out acquisition procedure using primed probe
Middle addition amplification enzyme.
Apply the technical scheme of the present invention, using primer amplification extend principle, using primer extend to sequence especially
The specific requirements of 3 ' end sequences are greater than in hybrid capture that base pair complementarity is to the specific requirements of sequence, with target gene
Opposite specific locations require highest 3 ' end as primer specificity in repeat region side conservative region or repeat region, if
Counting, there is the primed probe of detection label specifically to improve base in conjunction with Traditional liquid phase hybrid capture system as capture probe
Because of the capture rate of repeat region.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
The shortcomings that existing hybrid capture technology is can not to be designed probe to the high repetitive sequence in target gene region
Or the probe capture rate of design is not high.It is an object of the invention to improve the capture of high repeat region in target gene effect
Rate.
A kind of typical embodiment according to the present invention provides the spy that a kind of pair of Duplication region carries out hybrid capture
Needle.The probe is using specific locations having as 3 ' ends in target gene repeat region side conservative region or repeat region
Detect the primed probe of label.
Apply the technical scheme of the present invention, using primer amplification extend principle, using primer extend to sequence especially
The specific requirements of 3 ' end sequences are greater than in hybrid capture that base pair complementarity is to the specific requirements of sequence, with target gene
Opposite specific locations require highest 3 ' end as primer specificity in repeat region side conservative region or repeat region, if
Counting, there is the primed probe of detection label specifically to improve base in conjunction with Traditional liquid phase hybrid capture system as capture probe
Because of the capture rate of repeat region.
Preferably, 5 ' ends of primed probe have Illumina P5 joint sequence, and hybridization during subsequent applications is facilitated to catch
The progress obtained.Preferably, the biotin modification that detection is added labeled as the 5 ' ends in primed probe, during facilitating subsequent applications
The detection of hybrid capture signal.
Preferably, the length of primed probe is 63bp~73bp, preferably 68bp.This primed probe is of convenient length, and both wraps
Necessary joint sequence and specific 3 ' end sequences have been included, and has been unlikely to the too long difficulty and cost for increasing synthesis of length.
Preferably, primed probe can match special in target gene repeat region side conservative region or repeat region
The length of position is 5bp~20bp, preferably 10bp.In this case, 3 ' ends of primed probe can provide good
Specificity.
A kind of typical embodiment according to the present invention, target gene are ROS1 gene, and Duplication region is in No. 31
Containing subregion.
Preferably, the primed probe for including subregion progress hybrid capture to ROS1 gene 31 is as shown in table 1 below:
Table 1
Primed probe of the invention can be used alone specificity capture Duplication region, can also be with conventional polygenes
Capture probe pond be used in mixed way.
A kind of typical embodiment according to the present invention provides the side that a kind of pair of Duplication region carries out hybrid capture
Method.This method carries out hybrid capture to Duplication region using any of the above-described kind of probe.Preferably, hybrid capture uses liquid phase
Hybrid capture system carries out, and is carrying out that amplification enzyme is added in acquisition procedure using primed probe.
A kind of typical embodiment according to the present invention devises lung cancer, colorectal cancer target gene detection probe pond, packet
Included 16 genes exon region and it is partial include subregion, wherein be directed to target gene internal repeat region domain, design
Primer for specifically capturing, improves the capture rate of corresponding region.
The embodiment specific steps include:
1) analysis in target gene region.
The exonic DNA sequence that related 16 genes of lung cancer, colorectal cancer are obtained from NCBI, including ALK gene,
ROS1 gene includes destination region of the subregion as hybrid capture.
According to the DNA sequence dna of destination region, sequence of every 120bp as a probe compares tool using Blast, often
Probe compares human genome.Since gene extron regional sequence is relatively conservative, obtained similar sequences number is compared
Mesh (blast hit) is all smaller, can be used for designing the probe of hybrid capture.But No. 31 in ROS1 gene include sub-district
Domain, it was found that a large amount of repetitive sequence (table 2), these repetitive sequences can not be directly as capture probe.Due to ROS1 gene
It is Gene Fusion that Primary mutations form relevant to cancer, which occurs, and including in gene can occur on DNA level for Gene Fusion
Subregion, therefore improve to the capture rate in ROS1 gene intron region for improving the detection of Gene Fusion with important meaning
Justice.
No. 31 introne repeat region probes of table 2:ROS1 gene
2) design of specificity capture primer
According to the above analysis as a result, present embodiment is chosen according to the conservative region of repeat region flank and by Blast
Opposite specific base is held as primer 3 ' in repeat region, and modification and the Illumina of biotin is added at 5 ' ends of primer
P5 joint sequence.ROS1 the 31st includes 6840bp repetitive sequence in subregion and can design 20 articles of specificity captures primer (table 1).
3) capture and elution of target fragment are carried out using hybrid capture and special capture primer
Synthesize above 16 lung cancer, the capture probe of colorectal cancer related gene and specificity capture primer are mixed
It closes, is carrying out that amplification enzyme is added in acquisition procedure using probe, can be combined the special capture primer of design and purpose
The extension of segment.Since special primer end has biotin modification, the segment after subsequent hybrid capture, after extension
It can be together with the target fragment that hybrid capture obtains collected by the magnetic bead with streptavidin.Subsequent eluent can
That will not rinsed out by the segment that probe and special primer capture.
4.) PCR amplification of segment is captured
The target fragment both ends that probe or special primer capture either are hybridized all to have connected
Connector needed for the sequencing of Illumina platform, therefore expanded target fragment using general P5/P7 primer.
5) machine is sequenced on
The capture library of building is subjected to upper machine sequencing using Illumina microarray dataset, obtains raw sequencing data.
6) information analysis
Using hybrid capture information analysis process, the analysis of Quality Control parameter, including target area are carried out to raw sequencing data
Domain coverage, average sequencing depth, capture rate and homogeneity etc..
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment, are not expressly recited in following embodiment
The realization of ordinary skill in the art means can be used in step.
Embodiment 1
Sample information: cell line H1299DNA
Reagent and instrument see the table below 3:
Table 3
| Instrument title | Manufacturer | Model |
| SavantTMDNA SpeedVacTMInspissator | Thermo | DNA120-230 |
| Thermal cycler | BIO-RAD | T100TMThermal Cycler |
| 96 hole magnetic frames | Invitrogen | 12331D |
| Vortex oscillation instrument | Ancient cooking vessel sky source science and technology | VS-1 |
| 6 × 1.5mL of mini smart palm centrifuge | Ancient cooking vessel sky source science and technology | 0401061 |
| 2 × 8 × 0.2mL of mini smart palm centrifuge | Ancient cooking vessel sky source science and technology | 0401062 |
| Magnetic frame 1.5-2mL | ||
| Dry-type thermostat | Hangzhou Ao Sheng Instrument Ltd. | K30 |
| Reagent | Manufacturer | Article No. |
| 10 × washing buffer I | IDT | 1072273 |
| 10 × washing buffer II | IDT | 1072274 |
| 10 × washing buffer III | IDT | 1072275 |
| 10 × enhancing cleaning solution | IDT | 1072276 |
| 2 × hybridization buffer | IDT | 1072277 |
| Hybridization enhancers | IDT | 1072278 |
| 2 × magnetic bead cleaning solution | IDT | 1072279 |
| xGen Universal blockers | IDT | 1076154 |
| Dynabeads M-270streptavidin | Thermo | 65306 |
| Human cot-1DNA | Thermo | 15279-101 |
| KAPA HIFI hotstart ready mix | KAPA | KK2601 |
| Qubit dsDNA HS Assay kit | Thermo | Q32854 |
| Nuclease free water | Thermo | AM9930 |
| Dehydrated alcohol | Beijing Chemical Plant | Nothing |
| VAHTS DNA Clean Beads | Vazyme | N411-02 |
Operating process:
1.DNA is extracted: cell line H1299 cell line dna is extracted using regular growth system DNA extraction kit, after extraction
DNA it is quantitative using Qubit, take 200ng for capturing the building in preceding library.
2. library construction before capturing: cell line dna is broken into the segment of 200~300bp, is sequenced using universal genetic with text
Library kit, is carried out according to kit specification the building for capturing preceding library, and carries out purifying to library and Qubit is quantitative.
3. solution hybridization captures
The preparation of 3.1 capture probes
Prepare two parts of probes, middle probe 1 be according to lung cancer, 16 related genes of colorectal cancer (be respectively as follows: EGFR,
ALK, ROS1, MET, ERBB2, RET, NTRK1, KRAS, BRAF, PIK3CA, NRAS, AKT1, UGT1A1, KIT, PDGFRA and
TP53 the hybrid capture probe (commission IDT synthesis)) designed；Probe 2 is according to the special of No. 31 introne designs of ROS1 gene
Mixture of the primer (table 1, commission IDT synthesis) with probe 1 is captured, two parts of probes are diluted to working concentration respectively is
0.75nM is placed in ice chest.
3.2 take each 500ng in the preceding library of capture of two parts of preparations respectively, are named as sample 1 and sample 2, every part of sample difference
5ug Human Cot1DNA and 2ul tab closure agent is added, is vortexed and mixes.And it is dried in 70 DEG C or less, about 1.5~2h.
3.3 by the mixture dried in previous step be added 8.5 μ 2 × hybridization buffers of L, 2.7 μ L hybridization enhancers and
1.8 μ L nuclease free waters, are mixed by inversion and are being placed at room temperature for 5~10min；
3.4 are transferred to mixture in the PCR pipe of 0.2mL, 95 DEG C of incubation 10min；
3.5 at room temperature, and 4 μ L probes 1 (hybrid capture probe) are added in sample 1, and it is (miscellaneous that 4 μ L probes 2 are added in sample 2
Friendship capture probe+specifically capture primer)；It is vortexed and mixes, and brief centrifugation.Again in PCR instrument 65 DEG C be incubated for 4h (PCR instrument plus
Heat lid is set as 75 DEG C).
The preparation of 3.6 elution buffers
1 × elution buffer is shown in Table 4.
Table 4
Note: elution buffer will shift to an earlier date before preparing is being placed at room temperature for 2h.
The incubation of 3.7 buffers, parameter are shown in Table 5.
Table 5
| Solution | 1 × elution buffer | Operating temperature |
| Cleaning buffer solution I | 100μL | 65℃ |
| Cleaning buffer solution I | 200μL | Room temperature (15~25 DEG C) |
| Enhance cleaning solution | 400μL | 65℃ |
The preparation of 3.8 magnetic beads
A) Equilibrate is taken out from 4 DEG C of refrigeratorsM-270 magnetic bead, vortex 15s, is placed at room temperature
30min or so.
B) each 200 μ L EP manage in be added 100 μ L magnetic beads, magnetic frame be adsorbed to solution it is limpid after, it is careful with pipette tips
It draws and abandons supernatant；
C) 200 μ L 1 × magnetic bead cleaning solutions are added, and is vortexed and mixes 10s.Magnetic frame be adsorbed to solution it is limpid after, use rifle
Careful draw of head abandons supernatant；
D) step 3 is repeated；
E) be added 100 μ L 1 × magnetic bead cleaning solutions, magnetic frame be adsorbed to solution it is limpid after, discarding is carefully drawn with pipette tips
Supernatant.
3.9 Streptavidin MagneSpheres adsorb hybrid capture target area
The product of step 3.5 is transferred in the EP pipe in step 3.8-e, is mixed by inversion 10 times；(the lid on thermal cycler
Sub- temperature is set as 75 DEG C) 65 DEG C of incubation 45min are carried out, wherein every 12min vortex 3s.
3.10. Streptavidin MagneSphere is rinsed to remove unbonded DNA
A) 1 × cleaning buffer solution I of 65 DEG C of 100 μ L preheatings is added, is vortexed and mixes simultaneously brief centrifugation；
B) magnetic frame be adsorbed to solution it is limpid after, with pipette tips carefully draw abandon supernatant；
C) 200 μ L are added enhances cleaning solution, is mixed by inversion 10 times, and in 65 DEG C of incubation 5min；
D) magnetic frame be adsorbed to solution it is limpid after, with pipette tips carefully draw abandon supernatant；
E) step h and i are repeated.
F) 1 × cleaning buffer solution I that 200 μ L room temperature are incubated for is added, is vortexed and mixes 2min, and brief centrifugation；
G) magnetic frame be adsorbed to solution it is limpid after, with pipette tips carefully draw abandon supernatant；
H) 1 × cleaning buffer solution II that 200 μ L room temperature are incubated for is added, is vortexed and mixes 1min, and brief centrifugation；
I) magnetic frame be adsorbed to solution it is limpid after, with pipette tips carefully draw abandon supernatant；
J) 1 × cleaning buffer solution III that 200 μ L room temperature are incubated for is added, is vortexed and mixes 30s, and brief centrifugation；
K) magnetic frame be adsorbed to solution it is limpid after, with pipette tips carefully draw abandon supernatant.
L) upper step EP pipe is transferred on rack for test tube, and 20 μ L nuclease free waters is added, turn upside down mixing 10 times to be resuspended
Magnetic bead.
4. the PCR amplification after hybrid capture
4.1.PCR reaction reagent with the following table 6 of tabulating:
Table 6
Prepared PCR mixture is vortexed and is mixed, and brief centrifugation.
4.2 carry out PCR reaction according to the PCR response procedures in table 7:
Table 7
5. the purifying after amplification
5.1 are added 75 μ L (1.5 ×)In XP magnetic bead to each PCR amplification pipe, it is vortexed and mixes,
And room temperature is incubated for 10min；
5.2 magnetic frames adsorb 5min, after solution is limpid, is carefully drawn with pipette tips and abandon supernatant；
5.3, which are added 200 μ L, 80% ethyl alcohol, rinses DNA, and 30s is stood on magnetic frame, is drawn with pipette tips and abandons supernatant；
5.4 repeat previous step；
5.5 brief centrifugations are collected to ethyl alcohol to tube bottom, and exhaust tube bottom residual liquid, and room temperature dries 5~10min；
5.6 are added 22 μ L nuclease free waters, are vortexed and mix, are stored at room temperature 5min；
5.7 brief centrifugations are drawn in 20 μ L liquid to new EPR pipe；
6. library Quality Control
Using VAHTSTM Library Quantification Kit forKit carries out quantitative hybridization
The DNA concentration of capture detects library fragments using Bioptic Qseq100.
7. carrying out the sequencing in library using Illumina Nextseq550 sequenator.
8. using analysis of biological information process, initial data is analyzed.Analyze result such as the following table 8:
Table 8
It can be seen that the spy of the special capture primer comprising designing according to No. 31 intrones of ROS1 gene from the data of table 8
Needle 2 improves the capture rate of probe, especially ROS1 introne capture rate and is increased to 40% by the 10% of typical probe 1.
It can be seen from the above description that the above embodiments of the present invention realized the following chievements:
The method provided by the invention captured to Duplication region, can effectively improve the capture rate of probe.
It is had the advantage that compared to conventional hybridization catching method
1) the special capture primer that the present invention designs can have more high specific, Neng Gouti than standard hybridization capture probe
Height is to gene repeat sequence capture rate；
2) the special capture primer that the present invention designs can be used alone special capture repeat region, can also be with conventional more
The capture probe pond of gene is used in mixed way.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Co., Ltd, Tianjin promise standing grain medical test institute
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Claims (7)
1. the probe that a kind of pair of Duplication region carries out hybrid capture, which is characterized in that the probe is with target gene weight
The primed probe with detection label of the specific locations as 3 ' ends in multiple region side conservative region or repeat region；
5 ' ends of the primed probe have Illumina P5 joint sequence；
The biotin modification that the detection is added labeled as the 5 ' ends in the primed probe；
The length of the primed probe is 63bp~73bp；
The primed probe can match the specific locations in target gene repeat region side conservative region or repeat region
Length is 5bp~20bp.
2. probe according to claim 1, which is characterized in that the primed probe can be matched in target gene duplicate block
The length of specific locations is 10bp in domain side conservative region or repeat region.
3. probe according to claim 1, which is characterized in that the target gene is ROS1 gene, Duplication region
Subregion is included for No. 31.
4. probe according to claim 3, which is characterized in that include subregion to ROS1 gene 31 and carry out hybrid capture
Primed probe it is as shown in table 1 below:
Table 1
5. probe according to claim 1, which is characterized in that specificity capture gene weight is used alone in the primed probe
Multiple region, or be used in mixed way with conventional polygenic capture probe pond.
6. the method that a kind of pair of Duplication region carries out hybrid capture, which is characterized in that using as appointed in claim 1 to 5
Probe described in one carries out hybrid capture to Duplication region.
7. according to the method described in claim 6, it is characterized in that, the hybrid capture using solution hybridization capture system into
Row is carrying out that amplification enzyme is added in acquisition procedure using the primed probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811564939.5A CN109777858A (en) | 2018-12-20 | 2018-12-20 | The probe and method of hybrid capture are carried out to Duplication region |
Applications Claiming Priority (1)
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