CN108148891A - The probe preparation method of DNA of tumor cell injury repair related gene capture sequencing - Google Patents

Abstract

The invention discloses a kind of probe preparation method of DNA of tumor cell injury repair related gene capture sequencing, target gene includes 58 DNA of tumor cell injury repair related genes in this method；This method includes at least following steps：Step 1：Prepare the full exon region probe of 58 HRD genes；Step 2：Capture 58 target gene DNA；Step 3：Carry out elution program；Step 4：PCR reaction systems expand after carrying out capture target dna；Step 5：After amplified production magnetic beads for purifying, quantitative to 20ng/ul with qubit, packing preserves.The probe preparation method of the present invention highly effective can be solved at present for the polygenic Acquisition Detection of the homologous recombination repairs defect such as clinically breast cancer, oophoroma, cancer of pancreas and prostate cancer for 58 gene traps with reference to high-flux sequence platform.

Description

The probe preparation method of DNA of tumor cell injury repair related gene capture sequencing

Technical field

The mutation of homologous recombination repair dcc gene and mispairing gene repair after being damaged the present invention relates to a kind of DNA of tumor cell The probe preparation method of the capture sequencing of defect.Genetic risk available for breast cancer, oophoroma, prostate cancer and cancer of pancreas is commented Valency and targeted drug detection.

Background technology

The development of new-generation sequencing technology, the research for modern genetic group opens new situation, however genome is surveyed The cost of sequence and the complexity of analysis still allow researcher to feel more difficult, and the appearance of target sequence capture technique alleviates These problems.Target sequence capture sequencing is for the sequencing carried out after known specific gene group region design probe.It is outer aobvious Subgroup capture sequencing is then one of special case, it is specific to the research of whole exon regions in genome.More bases Because the sequencing of full extron is that high throughput is carried out after the full exon 1 domain dna of polygenes is captured and is enriched with using sequence capturing technology The genome analytical method of sequencing.Since it has to common and rare variation high sensitivity, exon 1 portion big absolutely can be found Point disease correlation variation and the advantages that the genome to about 1% is only needed to be sequenced, promotes the full extron sequencing of polygenes As the most effective strategy of Disease-causing gene of identification such as Mendel's disease disease, it is also employed for complex disease tumor susceptibility gene In research and clinical diagnosis.

Target sequence captures the specific fragment for referring to selectively detach or be enriched with by some way genome.From This definition sees that the PCR of target fragment is also one kind of target sequence capture, but this method flux is small, a current PCR Longest genomic DNA fragment is obtained no more than 50Kb, and needs special enzyme and special PCR conditions, it is expensive, surely Qualitative difference.Another catching method is according to the probe of nucleic acid molecules base complementrity, these probes are fixed on certain supports On, for detaching, genomic DNA is then interrupted, in addition hybridizing behind street corner with probe, is eluted as the DNA in hybridization, recycling target DNA fragmentation can directly build library and carry out DNA sequencing.

This method experimentation is simple, inexpensive.

Invention content

The purpose of the present invention is to provide a kind of probe preparations of DNA of tumor cell injury repair related gene capture sequencing Method, the capture probe of target gene in this method, be the target gene of biotin labeling specific probe group, the target Gene includes following 58 DNA of tumor cell injury repair related gene：RAD50、RAD51、RAD51D、RAD51C、RAD51B、 Rad52、BRIP1、BARD1、BAP1、WRN、FANCA、FANCD2、FAM175A、CHEK2、CHEK1、ATM、ATR、BRCA1、 BRCA2、NBN、PALB2、MRE11A、XRCC3、CDK12、c11orf30、CCNE1、FANCC、FANCI、POLQ、MMS22L、 RMI2、TP53、PTEN、STK11、PI3K、TSC1、TSC2、MLH1、MSH2、MSH6、PMS2、EPCAM、POLE、JAK1、JAK2、 NF1、NOTCH1、KRAS、MDM2、MDM4、DNMT3A、B2M、TS、ERCC2、ERCC6、MSH4、FANCM、CEP164；This method Including at least following steps：

Step 1：Prepare the full exon region probe of 58 HRD genes；

Step 2：Capture 58 target gene DNA；

Step 3：Carry out elution program；

Step 4：PCR reaction systems expand after carrying out capture target dna；

Step 5：After amplified production magnetic beads for purifying, quantitative to 20ng/ul with qubit, packing preserves.

Further, in the step 2, including at least as follows step by step：

Step 2.1：The chip of 12K difference oligonucleotides quantity is synthesized, the chip includes 200bp target areas and leads to 15 base ends：

5′-ATCGCACCAGCGTGTN120CACTGCGGCTCCTCA-3′；

Step 2.2：The oligonucleotide library of synthesis is dissolved in the low concentration TE solution of 500ul；

Step 2.3：A concentration of fmol ranks of each oligonucleotides, amplification obtain required concentration；

Step 2.4：PCR product is run with 5% glue, the PCR product of 230bp sizes is then recycled, is dissolved into 100ul water；

Step 2.5：Solution company is carried out by PCR, obtains the single-stranded oligonucleotide fragment with biotin.

Further, in the step 2.3,

Primer sequence is specially：

A 5′Biotin-CTGGGAATCGCACCAGCGTGT-3′

B 5′-CGTGGATGAGGAGCCGCAGTG-3′。

Further, in the step 2.3, amplification condition is：Prepare 3 50ul PCR reaction systems, template difference For 1ul, 2ul, 5ul；

PCR reaction conditions：95℃5min；95℃30S；60℃2min.

Further, in the step 3, including at least as follows step by step：

Step 3.1：Prepare following mixed liquor：The Adaptor Block and 6ul of 4ul 500ng DNA libraries, 2ul 200uM capture probe；

Step 3.2：In PCR instrument, 95 DEG C of 5min, 60 DEG C of 5min；

Step 3.3：Add in the 2X hybridization solutions of 60 DEG C of preheatings of 10ul；

Step 3.4：Hybridize for 24 hours for 60 DEG C in PCR instrument.

The solution have the advantages that：The probe preparation method of the present invention is measured for 58 gene traps with reference to high pass Sequence platform highly effective can solve homologous heavy for clinically breast cancer, oophoroma, cancer of pancreas and prostate cancer etc. at present Group repairs the polygenic Acquisition Detection of defect.

Specific embodiment

The probe preparation method of DNA of tumor cell injury repair related gene capture sequencing, this method include the following steps：

(1) total DNA in normal blood is extracted, extracts and interrupts DNA；

(2) end-filling, 3 ends add " A "；

(3) connector is connected and is purified；

(4) Pre-PCR is reacted and is purified；

(5) enriched DNA fragments probe hybridizes；

(6) magnetic capture target area domain dna；

(7) Pro-PCR is reacted and is purified.

The method that step (1) interrupts DNA is：

1. the genomic DNA of preparation is transferred to 0.6mL Maxy Clear Snaplock according to more than system In Microcentrifuge Tube, abundant mixing, of short duration centrifugation is placed on for use on ice；

2. opening ultrasound in advance interrupts instrument Bioruptor Pico, after SAPMAC method instrument temperature is down to 4 DEG C, arrange parameter ON 30s, OFF 30s are 1 cycle, are a wheel per 10cycles, carry out 3 wheels altogether, sample is placed on oscillator after every group Abundant mixing carries out next round and interrupts (mixing is more abundant, interrupts sample fragment size and more concentrates) after of short duration centrifugation；

3. 1 μ l samples is taken to carry out segment detection using Agilent2100 (DNA 1000Assay), rear sample is normally interrupted Main peak is detected about in 150bp-200bp.

Step (2) end-filling, 3 ends add the method for " A " to be：

1. in the sample cell after interrupting, by following table configuration reaction system (this operation carries out on ice chest)：

Component	Amount
		The double chain DNA fragment interrupted	50μl
End Repair＆A-Tailing buffer solutions	7μl
		End Repair&A-Tailing Enzyme Mix	3μl
Total volume	60μl

2. brief concussion centrifugation, PCR pipe is put back on ice chest, carries out lower step experiment immediately；

3. running PCR instrument program, PCR instrument parameter is set.Hot 85 DEG C of lid temperature setting；Heating module sets 20 DEG C, the time 30min；65 DEG C, time 30min；4℃∞；

4. PCR pipe is put into PCR instrument, program is run；Lower step coupled reaction is carried out immediately after the completion of program operation.

Simultaneously purification process is for the connector connection of step (3)；

1. adapter is diluted to suitable concentration by according to the form below in advance：

DNA50 microlitres of total amount of segment	Adapter stock concentration	Connector:Target gene ratio
			500ng	15μM	20:1

2. in the PCR pipe of above-mentioned step2 reactions, by following table configuration reaction system, (this operation is enterprising in ice chest Row)：

Component	Volume
		The sample terminated from step2 reactions	60μl
Connector (concentration as required)	5μl
		Seedless sour water	5μl
Connect buffer solution	30μl
		DNA ligase	10μl
Total volume	110μl

3. gently inhaling and beating mixing 6 times, avoid generating bubble, then of short duration centrifugation；

4. running PCR instrument program (it is required that being covered without heat), PCR instrument parameter is set, heating module sets 20 DEG C, time 15min. PCR pipe is put into PCR instrument, runs program；

It is 5. Agencourt AMPure XP magnetic beads is uniformly mixed spare by being shaken in advance to room temperature；

6. it in the PCR pipe carried out in step3 steps, is tested according to the purifying magnetic bead of 0.8X volumes, by following table Lattice configuration reaction system (this operation carries out on ice chest)：110 μ l of sample：Agencourt AMPure XP beads 88μl；

7. it gently inhales and beats mixing 6 times；

5-15min is incubated 8. being stored at room temperature, PCR pipe is placed in 3min on magnetic frame；

9. removing supernatant, PCR pipe continues to be placed on magnetic frame, and 200 μ l, 80% ethanol solutions are added in into PCR pipe, quiet Put 30s；

10. removing supernatant, then 200 μ l, 80% ethanol solutions are added in into PCR pipe, supernatant is thoroughly removed after standing 30s (removing bottom residual ethanol solution using 10 μ l pipettors)；

11. being stored at room temperature 3-5min, residual ethanol is made thoroughly to volatilize；

12. adding in the Nuclease free water of 22 μ l, PCR pipe from magnetic frame is removed, gently inhales and beats resuspension magnetic Pearl avoids generating bubble, is stored at room temperature 2min；

13. PCR pipe is placed in 2min on magnetic frame；

14. drawing 20 μ l supernatants with pipettor, it is transferred in new PCR pipe and (is placed on ice chest), in reaction tube subscript Remember sample number, prepare to react in next step.

The Pre-PCR reactions of step (4) and purification process：

1. in the pipe of step4 steps 10, by following table lattice configuration reaction system (this operation carries out on ice chest)：

Component	Quantity
		Sample from step4 steps 10	20μl
KAPA HiFi HotStart ReadyMix(2X)	25μl
		PE1.0(10μM)	2.5μl
PE2.0-indexX(10μM)	2.5μl
		Total amount	50μl

2. mixing is gently blown using pipettor, then of short duration centrifugation；

3. sample is placed in PCR instrument, start PCR programs, it is as follows：

4. the purifying of magnetic bead is carried out after the completion of program operation immediately.

5. adding in 50 μ l Agencourt AMPure XP magnetic beads into the PCR pipe after the reaction of step5, blown with pipettor Mixing is beaten, avoids generating bubble；

6. being incubated at room temperature 5-15min, PCR pipe is placed in 3min on magnetic frame；

7. removing supernatant, PCR pipe continues to be placed on magnetic frame, and 200 μ l, 80% ethanol solutions are added in into PCR pipe, quiet Put 30s；

8. removing supernatant, then 200 μ l, 80% ethanol solutions are added in into PCR pipe, thoroughly removing supernatant after standing 30s (makes Bottom residual ethanol solution is removed with 10 μ l pipettors)；

9. being stored at room temperature 5min, residual ethanol is made thoroughly to volatilize；

10. adding in the Nuclease free water of 30ul, centrifuge tube is removed from magnetic frame, using pipettor, gently Suction, which is beaten, is resuspended magnetic bead；

11. being stored at room temperature 2min, 200 μ l PCR pipes are placed in 2min on magnetic frame；

It (is placed on ice chest) 12. supernatant is transferred in 200 new μ l PCR pipes with pipettor, is marked on reaction tube Good catalogue number(Cat.No.) prepares to react in next step；

13. 1 μ l samples is taken to use3.0 Fluorometer (Qubit dsDNA HS Assay Kit) are into style of writing Library concentration mensuration records library concentration；

14. 1 μ l samples is taken to use 2100 Bioanalyzer system of Agilent (Agilent DNA 1000Kit) Library fragments length measurment is carried out, library length is about between 220-320bp.

The enriched DNA fragments probing procedure of step (5)：

1. ready sample and 58 gene trap probe mixtures are put into vacuum concentration centrifuge, PCR pipe is opened Lid starts centrifuge, opens vacuum pump switch, starts concentration (when can first be concentrated before concentration with the water of same volume Between test, calculate sample unit volume reduction time, confirm concentration speed, avoid when carrying out sample concentration, because concentrate when Between it is long cause over-drying, sample is caused to be lost)；

2. the sample drained is dissolved in again in 9 μ l Nuclease-free water, about 10 μ l of total volume, gently inhales and beat Mixing, of short duration centrifugation are placed on for use on ice；

3. 58 gene trap probe buffer solutions are placed in room temperature to melt, precipitation is had before not heating and is occurred, mixing postposition It is preheated in 65 DEG C of water-baths, (no precipitation and muddy object) takes 20 μ l, 58 gene trap probe buffer solutions after being completely dissolved；

4. being placed in 200 new μ lPCR pipes, pipe lid is covered, labeled as A, it is for use to continue to be placed in incubation in 65 DEG C of water-baths；

5. 5 μ l RNase Block and 2 μ l Probe is taken to be placed in 200 μ lPCR pipes, gently inhale and play mixing, of short duration centrifugation Be placed on it is for use on ice, labeled as C；

6. PCR instrument parameter is set, 100 DEG C of heat lid, 95 DEG C, 5min；65 DEG C, hold；

7. PCR pipe B is placed in PCR instrument, procedure above is run；

When 8.PCR instrument temperature is down to 65 DEG C, PCR pipe A is placed in PCR instrument and is incubated, cover PCR instrument heat lid；

9. after 5min, C being placed on PCR and is incubated, PCR instrument heat lid is covered；

10. after PCR pipe C is placed into PCR instrument 2min, pipettor is adjusted to 13 μ l, 13 μ l Hyb are drawn from PCR pipe A Buffer solution is moved in PCR pipe C, draws sample in whole PCR pipe B；

11. moving in PCR pipe C, gently inhale and make a call to 10 times, abundant mixing avoids generating a large amount of bubbles, and seal pipe lid covers PCR instrument heat lid, 65 DEG C are incubated overnight (16-24h).

The method of step (6) magnetic capture target area domain dna：

1. magnetic bead (Dynabeads MyOne Streptavidin T1magnetic beads) is vortexed from 4 DEG C of taking-ups Concussion is resuspended；

2. 50 μ l magnetic beads is taken to be placed in new PCR pipe, 1min on magnetic frame is placed in, removes supernatant；

3. removing PCR pipe from magnetic frame, 200 μ L Binding Buffer of addition, which gently inhale, beats mixing for several times, and magnetic is resuspended Pearl；

4. putting 1min on magnetic frame, supernatant is removed；

5. repeating step 3-4 twice, magnetic bead is cleaned altogether 3 times；

6. remove PCR pipe from magnetic frame, add in 200 μ L Binding Buffer and gently inhale and make a call to 6 resuspension magnetic beads and treat With；

7. hybrid product PCR pipe C is kept in PCR instrument, 200 μ L MyOne after the 6th step in step Step8 is resuspended T1magnetic beads are added in the product PCR pipe C after hybridization, are made a call to 6 mixings with pipettor suction, are placed in rotation blending instrument Upper room temperature combination 30min；

8. PCR pipe is placed in 2min on magnetic frame, supernatant is removed；

9. adding in the Wash Buffer 1 of 200 μ L into PCR pipe C, gently inhale and make a call to 6 mixings, be placed on rotation blending instrument 15min is cleaned, PCR pipe is put in 2min on magnetic frame, removes supernatant by then of short duration centrifugation；

10. adding in the Wash Buffer 2 of 65 DEG C of preheatings of 200 μ l, vortex mixing 5s is placed in ThermoMixer upper 65 DEG C 10min is incubated, 800 turns/min of rotating speed cleaned；

11. PCR pipe is put in 2min on magnetic frame, removes supernatant by of short duration centrifugation.Repetition is washed 2 times and is cleaned, 3 times altogether.Most It is primary afterwards thoroughly to remove except Wash Buffer2 (be removed with 10 μ l pipettors and be remained)；

12.PCR pipes continue to be placed on magnetic frame, and 200 μ l, 80% ethyl alcohol is added in into PCR pipe, are thoroughly moved after standing 30s Except ethanol solution (can be removed with 10 μ l pipettors and be remained), room temperature dries 2min；

13. adding in 30 μ L Nuclease-free water to PCR pipe, PCR pipe is removed from magnetic frame, gently inhales and makes a call to 6 Secondary resuspension magnetic bead is for use.

The method that step (7) Pro-PCR is reacted and purified：

1. needing to be enriched with DNA library after capture, Mix is prepared according to following table：

Component	Each sample volume
		Sample from step9 steps 7	30μl
Post PCR Buffer	18μl
		Post PCR Primer(25μM)	1μl
Post DNA Polymerase	1μl
		Total volume	50μl

2. pipettor is adjusted to 40 μ l, gently inhales and beat mixing 6 times, be subsequently placed in PCR instrument；

3. run PCR instrument program：

4.PCR terminates backward sample and adds in 55 μ l Agencourt AMPure XP magnetic beads, is gently inhaled and made a call to 6 times with pipettor Mixing；

5. being incubated at room temperature 5min, PCR pipe is placed in 3min on magnetic frame；

6. removing supernatant, PCR pipe continues to be placed on magnetic frame, adds in 200 μ l, 80% absolute ethyl alcohols, stands 30s；

7. removing supernatant, then 200 μ l, 80% absolute ethyl alcohols are added in into PCR pipe, thoroughly removing supernatant after standing 30 (can Bottom residual alcohol is removed with 10 μ l pipettors)；

8. it is placed at room temperature for 5min so that residual ethanol is thoroughly volatilized；

9. adding in 25 μ l Nuclease-free water, PCR pipe from magnetic frame is taken down, gently blows and beats mixing and magnetic is resuspended Pearl is placed at room temperature for 2min；

10. PCR pipe is placed in 2min on magnetic frame；

11. inhaling 23 μ l supernatants with pipettor is transferred to 1.5ml centrifuge tubes, sample message is marked；

12. 1 μ l libraries is taken to be quantified using Qubit dsDNA HS Assay Kit, library concentration is recorded, library is dense Degree is about in 1-10ng/ μ l；

13. take 1 μ l samples using Agilent 2100Bioanalyzer system (Agilent DNA 1000Kit) into Library fragment length of composing a piece of writing measures, and library length is about between 220-320bp；

14. it is sequenced using high-flux sequence platform.

Claims (5)

1. the probe preparation method of DNA of tumor cell injury repair related gene capture sequencing, it is characterised in that：Mesh in this method The capture probe of gene is marked, is the specific probe group of the target gene of biotin labeling, the target gene includes following 58 A DNA of tumor cell injury repair related gene：RAD50、RAD51、RAD51D、RAD51C、RAD51B、Rad52、BRIP1、 BARD1、BAP1、WRN、FANCA、FANCD2、FAM175A、CHEK2、CHEK1、ATM、ATR、BRCA1、BRCA2、NBN、 PALB2、MRE11A、XRCC3、CDK12、c11orf30、CCNE1、FANCC、FANCI、POLQ、MMS22L、RMI2、TP53、 PTEN、STK11、PI3K、TSC1、TSC2、MLH1、MSH2、MSH6、PMS2、EPCAM、POLE、JAK1、JAK2、NF1、 NOTCH1、KRAS、MDM2、MDM4、DNMT3A、B2M、TS、ERCC2、ERCC6、MSH4、FANCM、CEP164；This method is at least Include the following steps：

Step 1：Prepare the full exon region probe of 58 HRD genes；

Step 2：Capture 58 target gene DNA；

Step 3：Carry out elution program；

Step 4：PCR reaction systems expand after carrying out capture target dna；

Step 5：After amplified production magnetic beads for purifying, quantitative to 20ng/ul with qubit, packing preserves.

2. the probe preparation method of DNA of tumor cell injury repair related gene capture sequencing according to claim 1, It is characterized in that：In the step 2, including at least as follows step by step：

Step 2.1：The chip of 12K difference oligonucleotides quantity is synthesized, the chip includes 200bp target areas and general 15 base ends：

5′-ATCGCACCAGCGTGTN120CACTGCGGCTCCTCA-3′；

Step 2.2：The oligonucleotide library of synthesis is dissolved in the low concentration TE solution of 500ul；

Step 2.3：A concentration of fmol ranks of each oligonucleotides, amplification obtain required concentration；

Step 2.4：PCR product is run with 5% glue, the PCR product of 230bp sizes is then recycled, is dissolved into 100ul water；

Step 2.5：Solution company is carried out by PCR, obtains the single-stranded oligonucleotide fragment with biotin.

3. the probe preparation method of DNA of tumor cell injury repair related gene capture sequencing according to claim 2, It is characterized in that：In the step 2.3,

Primer sequence is specially：

A 5′Biotin-CTGGGAATCGCACCAGCGTGT-3′

B 5′-CGTGGATGAGGAGCCGCAGTG-3′。

4. the probe preparation method of DNA of tumor cell injury repair related gene capture sequencing according to claim 2, It is characterized in that：In the step 2.3, amplification condition is：3 50ul PCR reaction systems are prepared, template is respectively 1ul, 2ul, 5ul；

PCR reaction conditions：95℃5min；95℃30S；60℃2min.

5. the probe preparation method of DNA of tumor cell injury repair related gene capture sequencing according to claim 1, It is characterized in that：In the step 3, including at least as follows step by step：

Step 3.1：Prepare following mixed liquor：The Adaptor Block and 6ul of 4ul 500ng DNA libraries, 2ul 200uM capture probe；

Step 3.2：In PCR instrument, 95 DEG C of 5min, 60 DEG C of 5min；

Step 3.3：Add in the 2X hybridization solutions of 60 DEG C of preheatings of 10ul；

Step 3.4：Hybridize for 24 hours for 60 DEG C in PCR instrument.