A method for preparing a blocking reagent for a repetitive sequence using a multiple substitution amplification technique

Abstract

The present invention provides a method for preparing a blocking reagent for a repetitive sequence by using a multiple substitution amplification technique, the method comprising: amplifying genomic DNA by using a multiple substitution amplification technique; purifying the amplified product, breaking it into DNA fragments of 200-300 bp in length, and the resulting product is a blocking reagent for the repetitive sequence. The method of the present invention is used to prepare a blocking reagent for a repetitive sequence, which can avoid the disadvantages of the prior art method that placental DNA is not easy to obtain and the HAP column separation method is cumbersome, time-consuming and labor-intensive.

Description

Technical Field

[0001] The invention relates to a method for preparing a blocking reagent for a repeat sequence using a multiple substitution amplification technique, and belongs to the technical field of capture library construction. Background Art

[0002] Hybridization capture is one of the main technologies for capturing target regions in NGS. Two blocking reagents are usually used during the hybridization process to block the adaptor sequence and the repetitive sequence on the human genome respectively. Because the adaptor sequence and the repetitive sequence on the human genome may bind to the bases of part of the target library sequence complementarily, while capturing the target region library bound by the magnetic bead capture probe, this part of the library is also bound to the non-target library, and cannot be eluted during subsequent elution, resulting in excessive binding of the non-target library, resulting in reduced capture efficiency. Therefore, whether it is the blocking reagent for the adaptor sequence or the blocking reagent for the repetitive sequence on the human genome, any use of the blocking reagent incorrectly will significantly reduce the capture efficiency.

[0003] Currently, mainstream liquid phase capture products use two types of blocking reagents: universal blocking sequence and Cot-1 DNA; the function of the universal blocking sequence is to block the library connector and reduce connector hybridization; the function of Cot-1 DNA is to block the repetitive sequences on the human genome and reduce repetitive sequence hybridization.

[0004] The COT portion of human genomic DNA is mainly composed of rapidly annealing repetitive sequences. Interspersed repetitive sequences (IRS) such as SINEs (small interspersed repetitive sequences, such as Alu sequences) and LINEs (large interspersed repetitive sequences, such as L1 sequences) are widely distributed throughout the genome. Human COT DNA is prepared from human placental DNA by shearing, denaturation, and reannealing under conditions rich in these repetitive sequences.

[0005] The prior art “Preparation of human cot-1 DNA” (Ge Wenxin et al., Preparation of human cot-1 DNA [J] “Tumor”, May 1998, Vol. 18, No. 3 (127)) reported a method for separating and preparing cot-1 DNA using hydroxyapatite column chromatography (HAP column separation method). The method comprises the following steps: using ultrasound to break the obtained genomic DNA to a required size, calculating the required amount of the broken DNA according to the adsorption capacity of HAP and the amount of HAP loaded in the separation column, denaturing at 100°C for 10 minutes, quenching in an ice bath, calculating the DNA renaturation time t according to the formula cot=1=mol / L×Ts, renaturing cot-1DNA at 60°C for t seconds, immediately placing in an ice bath for 10 minutes after the renaturation, adding an equal volume of aqueous solution, adding the sample to the HAP separation column, adjusting the concentration of phosphate, eluting impurities such as free nucleotides and single-stranded nucleotides in sequence, and finally eluting the cot-1DNA elution peak, collecting the cot-1DNA, precipitating the collected cot-1DNA with two volumes of ethanol at room temperature, centrifuging at 8000 r / min at room temperature for 20 minutes, dissolving the obtained salt and cot-1DNA with appropriate water to obtain a co-precipitation mixture, and then using Sephadex G-50 desalting, collecting DNA peaks, adding 1 / 10 volume of 3M sodium acetate and two volumes of alcohol, centrifuging at 8000r / min and 4℃ for 20min, collecting the precipitate to obtain cot-1DNA. Although the preparation effect of the above-mentioned method for preparing cot-1DNA - HAP column separation method can meet the needs of hybridization, the preparation method is cumbersome, time-consuming and labor-intensive. At present, the commercial cot-1DNA of Invitrogen is a common commercial cot-1DNA, but the price is also relatively expensive. Summary of the invention

[0006] The main purpose of the present invention is to provide a new method for preparing a repetitive sequence blocking reagent.

[0007] To achieve the above object, the present invention provides a method for preparing a blocking reagent for a repetitive sequence, the method comprising:

[0008] The genomic DNA was amplified using multiple displacement amplification (MDA) technology;

[0009] After the amplified product is purified, it is sheared into DNA fragments of about 200-300 bp in length, and the resulting product is the blocking reagent for the repetitive sequence.

[0010] In the present invention, multiple displacement amplification (MDA) is used to amplify genomic DNA to prepare a blocking reagent for repeated sequences. MDA is a single-cell whole genome amplification technology developed in recent years. The technology is a chain displacement amplification technology based on the circular rolling amplification method. MDA uses phi29 DNA polymerase with high extension activity and hexameric random primers resistant to nuclease to amplify the genome under isothermal conditions. Compared with DOP-PCR and PEP-PCR, MDA has obvious advantages in both amplification efficiency and amplification credibility, and can uniformly amplify the whole genome without obvious deviation. MDA can amplify 20-30fg of product from 1-10 copies of genomic DNA.

[0011] The method of the present invention is used to prepare a blocking reagent for a repetitive sequence, thereby avoiding the disadvantages of the prior art methods that placental DNA is not easy to obtain and the HAP column separation method is cumbersome, time-consuming and labor-intensive.

[0012] According to a specific embodiment of the present invention, the method for preparing a blocking reagent for a repetitive sequence of the present invention further comprises a process of extracting genomic DNA.

[0013] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the extraction of genomic DNA can use existing techniques in the art, including using a commercial genomic extraction kit.

[0014] In some specific embodiments of the present invention, genomic DNA is extracted according to the following method:

[0015] When extracting genomic DNA, add nuclear lysis solution, proteinase K, and SDS to fully lyse the tissue, digest at 37°C-56°C for 12-16 hours; centrifuge at 3000-10000 rpm for 5-15 minutes to separate the DNA.

[0016] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the following primers are used in the process of amplifying genomic DNA using a multiple substitution amplification technique:

[0017] Primers for moderately repetitive sequences and / or highly repetitive sequences; or

[0018] The Cot-1 DNA was fragmented and used as random primers.

[0019] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the primers for the moderately repetitive sequence and / or the highly repetitive sequence may be selected from one or more of Alu, Kpn, and LINE. Preferably, the primers for the moderately repetitive sequence and / or the highly repetitive sequence include at least one of each of the Alu, Kpn, and LINE families, that is, at least include at least one of the Alu family, at least one of the Kpn family, and at least one (pair) of the LINE family.

[0020] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, when the Cot-1 DNA is broken and used as a random primer, the Cot-1 DNA is broken into small fragments of about 20-40 bp.

[0021] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the process of amplifying genomic DNA using a multiple substitution amplification technique is performed according to the following operations:

[0022] The sample is lysed, neutralized and centrifuged, and amplification mixture is added to perform PCR amplification. Preferably, during amplification, the PCR instrument is set at 30°C for 8 hours, 65°C for 5 minutes, and the heated cover is 70°C.

[0023] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the lysis solution formula used for lysis includes: per 1 reaction volume, lysis buffer 1.15 μl, DTT 0.35 μl, NF water 1.5 μl.

[0024] According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for a repetitive sequence of the present invention, the amplification mixture formula includes: per 1 reaction volume, 37 μl of amplification buffer, 2 μl of DNA polymerase, 3 μl of primers, and 0.5 μl of enhancer.

[0025] Unless otherwise specified, the reagent materials used in the present invention, such as buffers, polymerases, enhancers, etc., can be conventional reagent materials in the art.

[0026] In the method for preparing a blocking reagent for a repetitive sequence of the present invention, when the amplified product is purified and then interrupted, it can be performed according to conventional operations in the relevant field or the instructions of the instrument manufacturer.

[0027] According to a specific embodiment of the present invention, the method for preparing a blocking reagent for a repetitive sequence of the present invention further comprises a process of purifying, recovering and quality controlling the disruption product.

[0028] It should be noted that the length of the shearing described in the present invention, for example, "slicing into DNA fragments of about 200-300 bp in length", means that more than 80% of the DNA fragments obtained by shearing have a length within this range. Similarly, "slicing into small fragments of about 20-40 bp" means that more than 80% of the small fragments in the shearing products have a length of 20-40 bp.

[0029] The method for preparing a blocking reagent for a repetitive sequence of the present invention can achieve the same blocking effect as the commonly used COT1 DNA, and the preparation method is convenient and simple. DETAILED DESCRIPTION

[0030] The following describes the implementation process of the present invention and the beneficial effects produced by the specific examples in detail, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention. In the examples, the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or are operated according to the conditions recommended by the instrument manufacturer.

[0031] Example 1

[0032] This embodiment provides an MDA amplification method for preparing a repetitive sequence blocking reagent, and the main operations include:

[0033] Step 1: Extract genomic DNA

[0034] 1. In this example, the following steps were used to extract genomic DNA.

[0035] Take 25-50mg of fresh animal tissue, add 600μl of Lysis Buffer, and mix thoroughly by inversion. Add 10μl of Proteinase K (concentration is 10mg / ml) and mix well. Digest in a water bath at 56℃ for 12-16 hours, and centrifuge at 4700rpm for 10min to separate DNA and protein and other impurities. Since sufficient digestion time is given, the DNA has been well digested at this time, and the step of DNA purification can be avoided, which not only saves part of the reagents for DNA purification, but also avoids the loss of DNA in the purification process.

[0036] Step 2: Design and synthesize primers for amplifying highly repetitive sequences (such as Alu, Kpn, LINE, etc.)

[0037] 1. Primer sequences are as follows

[0038]

[0039] The above primers were synthesized using DSL method.

[0040] Step 3: MDA amplification reaction

[0041] 1. Sample Lysis

[0042] 1) The lysis buffer formula is as follows:

[0043] Reagents Dosage (1 reactions) Lysis buffer 1.15μl DTT 0.35μl NF Water 1.5μl

[0044] 2) Add 3 μl lysis buffer to 4 μl sample (gDNA ≥ 25 pg), shake and mix, and centrifuge.

[0045] 3) Lyse at 65°C for 10 min in a PCR machine with a heated cover at 70°C.

[0046] 2. Neutralization

[0047] Take 3μl Stop Solution and add it to the lysis mixture, shake and mix, and centrifuge.

[0048] 3. Amplification

[0049] 1) The formula of the amplification mixture is as follows:

[0050] Reagents Dosage (1 reactions) Amplification buffer 37μl DNA polymerase 2μl Primers 3μl enhancer 0.5μl

[0051] 2) Take 42.5 μl of amplification mixture and add it to the sample, with a total volume of 52.5 μl, and shake to mix.

[0052] 3) Set the PCR instrument to 30°C for 8 hours; 65°C for 5 minutes; and heat cover at 70°C

[0053] 4. MDA product purification

[0054] Use column purification, Kangwei Century CW2301M is recommended, and the specific operation is detailed in the instruction manual.

[0055] Step 4: MDA amplification product fragmentation

[0056] The following are the steps for Covaris M220 DNA Shearing (follow the manufacturer's instructions):

[0057] Power on inspection

[0058] 1) Check whether the line connection between the computer fixed on the top of the machine and the machine is proper.

[0059] 2) Make sure the Drip Tray is placed under the machine.

[0060] 3) Insert the operating tube holder (Tube Holder)

[0061] Water bath setup

[0062] 1) Pull up the sliding weight on the top of the tube holder and rotate it 90°.

[0063] 2) Use the wash bottle provided to add about 15 ml of distilled or deionized water to the center of the bracket. It is best if the water level reaches or exceeds the "RUN" marker.

[0064] Sample placement

[0065] 1) Pull up the sliding weight on the top of the tube holder and rotate it 90°.

[0066] 2) Place the sample tube, rotate and lower the sliding weight to press the sample tube, and then close the safety door.

[0067] 3) Power on: first turn on the instrument host, then turn on the computer and software.

[0068] Parameter settings

[0069] 1) Click Method in the Run interface of the software. The edited Method information will be displayed in the drop-down menu. You can select a Method, click New to create a new one, or select an existing method in the list and click Edit to edit it.

[0070] 2) Set relevant parameters according to the length of the target fragment to be interrupted. The specific parameters can be referred to as follows:

[0071] MicroTUBE AFA Fiber Screw-Cap-50ul sample-from 150bp to 800bp.

[0072] Target BP(Peak) 150 200 250 300 400 450 500 800 Peak Incident Power(W) 50 50 50 50 50 50 50 50 Duty Factor 20％ 20％ 20％ 20％ 20％ 20％ 20％ 20％ Cycles per Burst 200 200 200 200 200 200 200 200 Treatment Time(s) 375 175 120 80 50 40 32 25 Temperature(℃) 20 20 20 20 20 20 20 20 Sample volume (ul) 50 50 50 50 50 50 50 50

[0073] MicroTUBE AFA Fiber Snap-Cap-130ul sample-from 150bp to 1500bp.

[0074] Target BP(Peak) 150 200 300 400 500 800 1000 1500 Peak Incident Power(W) 50 50 50 50 50 50 50 50 Duty Factor 20％ 20％ 20％ 10％ 10％ 5％ 5％ 2％ Cycles per Burst 200 200 200 200 200 200 200 200 Treatment Time(s) 350 160 75 90 60 80 60 25 Temperature(℃) 20 20 20 20 20 20 20 20 Sample volume (ul) 130 130 130 130 130 130 130 130

[0075] Run the program

[0076] Click the "Run" button on the Run interface to run the program.

[0077] Shut down software and machines

[0078] 1) Empty the water bath using the syringe provided; if there is water in the drip tray, pour it out and wipe it dry with absorbent paper.

[0079] 2) Close the software, then the instrument host, and finally the computer.

[0080] Step 5: Purification, recovery and quality control of the interruption product

[0081] Yikang magnetic beads DNA purification and recovery reagent (Cat. No. YK003) was used for selective recovery of DNA fragments.

[0082] Note: Selective recovery of DNA fragments is an optional step. If the starting sample volume is less than 50 ng, it is not recommended to perform selective recovery of DNA fragments. You can refer to the instructions and directly purify the DNA fragments. In addition, when constructing DNA libraries of different sizes, the amount of magnetic beads used for selective recovery of DNA fragments is different. The specific amount of magnetic beads can be referred to as follows (if using magnetic beads from manufacturers other than Yikang, you need to find the optimal amount of magnetic beads by yourself).

[0083]

[0084] 1) Yield: ≥25μg before purification, ≥12μg after purification.

[0085] 2) Fragment size: Before purification, more than 80% of the product fragments were within the range of 200-300 bp in length by electrophoresis.

[0086] Example 2

[0087] Step 1: Extract genomic DNA

[0088] Refer to the genomic DNA extraction method in step 1 of Example 1.

[0089] Step 2: Cot-1 DNA fragmentation

[0090] Referring to the shearing method in step 4 of Example 1, Cot-1 DNA was sheared into small fragments of about 30 bp (about 80% or more of the actual sheared products were within the range of 25 bp-35 bp).

[0091] Step 3: MDA amplification reaction

[0092] MDA amplification was performed using genomic DNA as a template and the fragmented Cot-1 DNA as a primer. The specific operation steps were as shown in step 3 of Example 1. Finally, the MDA amplification product was purified and quantified.

[0093] Column purification was used, and Kangwei Century CW2301M was recommended. For detailed operation, please refer to the instruction manual. MDA amplification products were quantified using Qubit.

[0094] Step 4: MDA amplification product fragmentation

[0095] Referring to the interruption method in step 4 of Example 1, the MDA amplification product was fragmented.

[0096] Step 5: Purification, recovery and quality control of the interruption product.

[0097] Yikang magnetic beads DNA purification and recovery reagent (Cat. No. YK003) was used for selective recovery of DNA fragments. The recovered products were quantified using Qubit.

[0098] Example 3

[0099] This example verifies the effects of the blocking reagents prepared by the two methods in the above examples:

[0100] Targeted library preparation and sequencing:

[0101] 1. DNA extraction: Use the Kangwei Century Fixed Tissue Genomic DNA Extraction Kit to extract FFPE sample genomic DNA. Refer to the instruction manual for specific operations.

[0102] 2. Library construction: Use Cosmed NGS Fast DNA Library Prep Set for Illumina to construct the second-generation sequencing library. Refer to the instruction manual for specific operations.

[0103] 3. Hybridization, capture and elution of the library.

[0104] Take 3 1ug of the same constructed library into a new centrifuge tube, block L1 with the blocking reagent prepared in Example 1, block L2 with the blocking reagent prepared in Example 2, block L3 with COT1 DNA, and add the remaining components as follows:

[0105] Reagents Dosage library 1ug Repeat Blocking Reagent / COT1 DNA 5ul Universal Closing Sequence 1ul

[0106] Concentrate using a vacuum concentrator. For each concentrated library, add the remaining components as follows.

[0107] Reagents Volume(ul) 2X Hybridization Buffer 8.5 Hybridization Buffer Enhancer 2.7 Nuclease-free water 1.8

[0108] After gently vortexing to mix, let it stand at room temperature for 5 minutes, transfer it to a new PCR tube, and incubate it on a PCR instrument at 95°C for 10 minutes (hot cover temperature 105°C). Immediately after taking it out, add 4ul of capture probe and incubate it at 50°C overnight.

[0109] Elution: Elution was performed using IDT's elution reagents and methods.

[0110] 4. After PCR amplification and purification, the product is sequenced on an NGS machine.

[0111] Prepare the PCR system according to the following reagents:

[0112] Reagents volume Eluted DNA library 30μL PCR Buffer 18μL PCR Primer (25μM, for ILM) 1μL PCR Polymerase 1μL Total volume 50ul

[0113] The prepared PCR reaction system is placed in a PCR instrument for amplification. The reaction procedure is as follows:

[0114]

[0115] After the PCR reaction, 1X Agencourt AMPure XP magnetic beads were used for purification and 25 μl Nuclease-free water was used for elution (the library concentration was about 1-20 ng / μl). Three captured libraries were taken for Illumina sequencing, and the sequencing data volume was 200M. The data analysis results are as follows.

[0116] Library Name Data volume Capture efficiency Effective depth of target area L1 200M 85％ 1950X L2 200M 80％ 2103X L3 200M 79％ 2040X

[0117] The same sample and the same capture operation, the blocking reagent prepared by the patented method can achieve a blocking efficiency close to that of the commercially available COT1 DNA, proving that the two preparation methods are feasible.

[0118] The above description is only a specific implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Suzhou Yikang Medical Testing Co., Ltd. <120> A method for preparing a blocking reagent for a repetitive sequence using a multiple substitution amplification technique <130> GAI21CN4210 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 1 cctgaggtca ggagttcgag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 2 cccgagtagc tgggattaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 3 gggatctgca cccagaaaac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 4 aaggaccagt ctctagcgga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 5 gacaaatgct tcacacgggg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 6 gaaatggccg aggcgaaaac 20

Claims

1. A method for preparing a blocking reagent for a repetitive sequence, the method comprising: The genomic DNA was amplified using multiple substitution amplification technology. After the amplified product is purified, it is sheared into DNA fragments of 200-300 bp in length, and the resulting product is the blocking reagent for the repeat sequence; The method also includes a process of extracting genomic DNA, and the genomic DNA is extracted according to the following method: When extracting genomic DNA, add nuclear lysis buffer, proteinase K, and SDS to fully lyse the tissue, digest at 37℃-56℃ for 12-16 hours; centrifuge at 3000-10000rpm for 5-15min to separate the DNA; The process of amplifying genomic DNA using multiple substitution amplification technology is carried out as follows: The samples were lysed, neutralized, centrifuged, and amplification mixture was added for PCR amplification; During amplification, the PCR instrument was set at 30°C for 8 h; 65°C for 5 min; and the heated cover was set at 70°C. The following primers were used in the amplification of genomic DNA using the multiple displacement amplification technique: Primers for moderately repetitive sequences and / or highly repetitive sequences; or The Cot-1 DNA was fragmented and used as a random primer; The primers for the moderately repetitive sequence and / or the highly repetitive sequence include at least one of Alu, Kpn, and LINE family, the primers for Alu are shown in SEQ ID No. 1 and SEQ ID No. 2, the primers for Kpn are shown in SEQ ID No. 3 and SEQ ID No. 4, and the primers for LINE family are shown in SEQ ID No. 5 and SEQ ID No. 6; When Cot-1 DNA is fragmented and used as a random primer, the Cot-1 DNA is fragmented into small fragments of 20-40 bp.

2. The method according to claim 1, wherein: The lysis solution formula used for lysis includes: per 1 reaction volume, lysis buffer 1.15μl, DTT 0.35μl, NF water 1.5μl; The formula of the amplification mixture includes: per 1 reaction volume, 37 μl of amplification buffer, 2 μl of DNA polymerase, 3 μl of primers, and 0.5 μl of enhancer.

3. The method according to claim 1, further comprising a process of purifying, recovering and quality controlling the fragmentation product.

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