CN113444776A - Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology - Google Patents
Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology Download PDFInfo
- Publication number
- CN113444776A CN113444776A CN202110778770.9A CN202110778770A CN113444776A CN 113444776 A CN113444776 A CN 113444776A CN 202110778770 A CN202110778770 A CN 202110778770A CN 113444776 A CN113444776 A CN 113444776A
- Authority
- CN
- China
- Prior art keywords
- amplification
- dna
- blocking reagent
- primers
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 66
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 48
- 230000003321 amplification Effects 0.000 title claims abstract description 46
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 46
- 230000000903 blocking effect Effects 0.000 title claims abstract description 42
- 230000003252 repetitive effect Effects 0.000 title claims abstract description 36
- 238000005516 engineering process Methods 0.000 title claims abstract description 11
- 238000006467 substitution reaction Methods 0.000 title claims abstract description 7
- 239000012634 fragment Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 7
- 238000006073 displacement reaction Methods 0.000 claims description 7
- 230000009089 cytolysis Effects 0.000 claims description 5
- 239000003623 enhancer Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 239000012139 lysis buffer Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 230000003472 neutralizing effect Effects 0.000 claims description 2
- 230000002934 lysing effect Effects 0.000 claims 2
- 238000000926 separation method Methods 0.000 abstract description 6
- 230000007547 defect Effects 0.000 abstract description 2
- 210000002826 placenta Anatomy 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 50
- 239000000047 product Substances 0.000 description 20
- 239000000523 sample Substances 0.000 description 16
- 238000000746 purification Methods 0.000 description 14
- 239000011324 bead Substances 0.000 description 8
- 238000010828 elution Methods 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000007789 sealing Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 3
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 description 3
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 101710184482 Peroxisomal carnitine O-octanoyltransferase Proteins 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 108091023043 Alu Element Proteins 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101001055091 Homo sapiens Mitogen-activated protein kinase kinase kinase 8 Proteins 0.000 description 1
- 101000599612 Homo sapiens Peroxisomal carnitine O-octanoyltransferase Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 108091027568 Single-stranded nucleotide Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000013412 genome amplification Methods 0.000 description 1
- 102000049920 human MAP3K8 Human genes 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- -1 polymerases Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention provides a method for preparing a blocking reagent of a repetitive sequence by a multiple substitution amplification technology, which comprises the following steps: amplifying the genome DNA by adopting a multiple substitution amplification technology; after purifying the amplification product, breaking the DNA fragment with the length of 200-300bp, and obtaining the product which is the blocking reagent of the repetitive sequence. The method for preparing the blocking reagent of the repetitive sequence can overcome the defects that the placenta DNA is not easy to obtain and the HAP column separation method is complicated, time-consuming and labor-consuming in the prior art.
Description
Technical Field
The invention relates to a method for preparing a blocking reagent of a repetitive sequence by a multiple substitution amplification technology, belonging to the technical field of capture and library construction.
Background
Hybrid capture is one of the main technologies for capturing target regions in NGS, and two blocking reagents are usually used to block the sequence of the adaptor and the repetitive sequence on the human genome, respectively, in the hybridization process. Because the sequence of the adaptor and the repetitive sequence on the human genome may be in base complementary binding with a part of the target library sequence, when the target region library bound by the probe is captured by the magnetic bead, the part of the library is bound on the non-target library, and the non-target library cannot be eluted in the subsequent elution, so that the binding of the non-target library is excessive, and the capture efficiency is reduced. Therefore, whether the blocking reagent of the adaptor sequence or the blocking reagent of the repetitive sequence on the human genome, the capture efficiency is remarkably reduced when any one of the blocking reagents is used in error.
Currently, two types of blocking reagents are used in mainstream liquid phase capture products: universal blocking sequence and Cot-1 DNA; the universal blocking sequence is used for blocking the library joints and reducing joint hybridization; the Cot-1DNA has the function of blocking repetitive sequences on a human genome and reducing repetitive sequence hybridization.
The COT portion of human genomic DNA consists mainly of rapidly annealing repeats. SINE (small interspersed repeats, such as Alu sequences) and LINE (large interspersed repeats, 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.
Prior Art "preparation of human cot-1 DNA" (Wagnen et al, preparation of human cot-1DNA [ J ] "tumor (1998, 5 months, 18, 3 rd (127)) reported a method for separating and preparing cot-1DNA by hydroxyapatite column chromatography (HAP column separation). Breaking the obtained genome DNA to a required size by using ultrasonic waves, calculating the quantity of the broken DNA required according to the adsorption capacity of the HAP and the quantity of the HAP filled in a separation column, denaturing at 100 ℃ for 10min, quenching in an ice bath, calculating DNA renaturation time t according to a formula cot which is 1 mol/L multiplied by Ts, renaturing at 60 ℃ cot-1DNA for t seconds, immediately placing in the ice bath for 10min after renaturation is finished, adding an isovolumetric aqueous solution, adding a sample to the HAP separation column, sequentially eluting impurities such as free nucleotide and single-stranded nucleotide by adjusting the concentration of phosphate, finally eluting a cot-1DNA elution peak, collecting, precipitating the collected cot-1DNA by using ethanol with twice the volume at room temperature, centrifuging at 8000r/min for 20min at room temperature, dissolving the obtained salt and a coprecipitation mixture obtained by the cot-1DNA by using proper water, then desalting with Sephadex G-50, collecting DNA peak, adding 1/10 volume of 3M sodium acetate, and twice volume of ethanol, centrifuging at 4 deg.C for 20min, and collecting precipitate to obtain cot-1 DNA. The HAP column separation method, which is a method for preparing cot-1DNA, is complicated in preparation process, time-consuming and labor-consuming, although the preparation effect can meet the requirement of hybridization. Currently, the commercial cot-1DNA from Invitrogen is a common commercial cot-1DNA, but the price is also expensive.
Disclosure of Invention
The invention mainly aims to provide a novel method for preparing a repetitive sequence blocking reagent.
To achieve the above object, the present invention provides a method for preparing a blocking reagent for repetitive sequences, the method comprising:
amplifying the genome DNA by adopting a multiple displacement amplification technology (MDA);
after purifying the amplification product, breaking the DNA fragment with the length of about 200-300bp, and obtaining the product which is the blocking reagent of the repetitive sequence.
In the present invention, genomic DNA is amplified using Multiple Displacement Amplification (MDA) to prepare blocking reagents for repetitive sequences. MDA is a single-cell whole genome amplification technology developed in recent years, and is a strand displacement amplification technology created based on a circular rolling amplification method, and the MDA utilizes phi29DNA polymerase with high extension activity and an exonuclease-resistant hexamer random primer to amplify a genome under isothermal conditions. Compared with DOP-PCR and PEP-PCR, MDA has obvious advantages in amplification efficiency and amplification reliability, and can uniformly amplify the whole genome without obvious deviation. MDA can amplify 20-30fg product from 1-10 copies of genomic DNA.
The method for preparing the blocking reagent of the repetitive sequence can overcome the defects that the placenta DNA is not easy to obtain and the HAP column separation method is complicated, time-consuming and labor-consuming in the prior art.
According to a specific embodiment of the present invention, the method for preparing a blocking reagent for repetitive sequences of the present invention further comprises a process for extracting genomic DNA.
According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for repetitive sequences of the present invention, the extraction of genomic DNA can be performed using the existing techniques in the art, including the use of a commercially available genomic extraction kit.
In some embodiments of the invention, genomic DNA is extracted as follows:
when extracting genome DNA, adding nuclear lysis solution and protease K, SDS to fully crack tissues, digesting for 12-16 hours at 37-56 ℃; DNA was separated by centrifugation at 3000 and 10000rpm for 5-15 min.
According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for repeated sequences of the present invention, the following primers are used in the amplification of genomic DNA by the multiple displacement amplification technique:
primers of medium and/or high repeat sequence; or
The Cot-1DNA was broken and used as a random primer.
According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for repetitive sequences of the present invention, the primer for medium repetitive sequences and/or high repetitive sequences may be selected from one or more of Alu, Kpn, LINE. Preferably, the primer of the medium and/or high repeating sequence comprises at least one of Alu, Kpn, LINE family, i.e. at least one of Alu family, Kpn family and LINE family (pairs).
According to a specific embodiment of the invention, in the method for preparing blocking reagent for repetitive sequences, when Cot-1DNA is broken and used as a random primer, Cot-1DNA is broken to a small fragment of about 20-40 bp.
According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for repetitive sequences of the present invention, the genomic DNA is amplified by the multiple displacement amplification technique according to the following operations:
and (3) cracking the sample, neutralizing, centrifuging, adding the amplification mixed solution, and performing PCR amplification. Preferably, during amplification, the temperature in the PCR instrument is set to be 30 ℃ for 8 h; 5min at 65 ℃; the hot lid was 70 ℃.
According to a specific embodiment of the present invention, in the method for preparing a blocking reagent for repetitive sequences of the present invention, the lysis solution used for the lysis comprises: for each 1reaction volume, 1.15. mu.l lysis buffer, 0.35. mu.l DTT, 1.5. mu.l NF water.
According to a specific embodiment of the present invention, in the method for preparing blocking reagent for repetitive sequences of the present invention, the formulation of the amplification mixture comprises: for each 1reaction volume, 37. mu.l of amplification buffer, 2. mu.l of DNA polymerase, 3. mu.l of primers, 0.5. mu.l of enhancer.
Unless otherwise noted, the reagent materials used in the present invention, such as buffers, polymerases, enhancers, etc., may be those conventionally used in the art.
In the method for preparing a blocking reagent for a repetitive sequence of the present invention, when the amplification product is interrupted after purification, the procedure may be performed according to the instructions of the conventional procedures or instrument manufacturers in the art.
According to the specific embodiment of the invention, the method for preparing the blocking reagent of the repeated sequence further comprises the processes of purifying, recovering and controlling the quality of the interrupting product.
The term "disrupted length" as used herein, for example, "a DNA fragment having a length of about 200 bp to about 300 bp" means that 80% or more of the fragments obtained by disruption have a length within the above length range. Similarly, "fragmented to a small fragment of about 20-40 bp" means fragmented to a small fragment of more than 80% of the product, and preferably 20-40bp in length.
According to the method for preparing the blocking reagent of the repetitive sequence, the blocking reagent of the repetitive sequence prepared by the method can achieve the same blocking effect with the common COT1DNA, and meanwhile, the preparation method is convenient and simple.
Detailed Description
The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure. In the examples, the experimental methods without specifying the specific conditions were conventional methods and conventional conditions well known in the art, or were operated according to the conditions suggested by the instrument manufacturer.
Example 1
This example provides a method for preparing a blocking reagent for repeated sequences by using an MDA amplification method, which comprises the following steps:
the method comprises the following steps: extraction of genomic DNA
1. In this example, the following procedure was used to extract genomic DNA.
Fresh animal tissue 25-50mg was added to 600. mu.l of lysine Buffer, and mixed well by inversion. Add 10. mu.l of Proteinase K (10 mg/ml) and mix well. Digesting in water bath at 56 deg.C for 12-16 hr, and centrifuging at 4700rpm for 10min to separate DNA and protein. Because sufficient digestion time is given, the DNA is well digested at the moment, the step of purifying the DNA can be omitted, a part of reagents for purifying the DNA are saved, and the loss of the extracted DNA in the purification process can be avoided.
Step two: design of primers for synthetic amplification of highly repetitive sequences (e.g., Alu, Kpn, LINE, etc.)
1. The primer sequences are shown below
The above primers were synthesized by DSL method.
Step three: MDA amplification reaction
1. Sample lysis
1) The lysate formula is as follows:
| reagent | Dosage (1 interactions) |
| Lysis buffer | 1.15μl |
| DTT | 0.35μl |
| NF water | 1.5μl |
2) Mu.l of sample (gDNA is more than or equal to 25pg), adding 3. mu.l of lysate, shaking, mixing uniformly and centrifuging.
3) The PCR instrument was lysed for 10min at 65 ℃ with a hot lid of 70 ℃.
2. Neutralization
Adding 3 μ l of Stop Solution into the cracking mixture, shaking, mixing, and centrifuging
3. Amplification of
1) The formula of the amplification mixed solution is as follows:
| reagent | Dosage (1 interactions) |
| Amplification buffer | 37μl |
| DNA polymerase | 2μl |
| Primer and method for producing the same | 3μl |
| enhancer | 0.5μl |
2) Adding 42.5 μ l of the amplification mixture into the sample, shaking and mixing to obtain a mixture with a total volume of 52.5 μ l
3) Setting the temperature in a PCR instrument at 30 ℃ for 8 h; 5min at 65 ℃; thermal cover 70 deg.C
MDA product purification
The column type purification is adopted, the product is recommended to be century CW2301M, and the specific operation is described in the specification.
Step four: fragmentation of MDA amplification product
The following is the Covaris M220 DNA cleavage step (as per the manufacturer's instructions):
boot check
1) Checking whether the computer fixed on the top of the machine is properly connected with the circuit of the machine.
2) A confirmation water Tray (drop Tray) was placed under the machine.
3) Insertion operating Tube support (Tube Holder)
Water bath device
1) The Sliding Weight (Sliding Weight) on top of the handling Tube Holder (Tube Holder) was pulled up and rotated 90 ℃.
2) Approximately 15ml of distilled or deionized water was added to the center of the stent using a water random bottle (wash bottle), preferably at or above the RUN marker ("RUN" marker).
Sample placement
1) The Sliding Weight (Sliding Weight) on top of the handling Tube Holder (Tube Holder) was pulled up and rotated 90 ℃.
2) The sample tube is put in, the sliding weight is rotated and put down to press the sample tube, and then the safety door is closed.
3) Starting up: the instrument host is started first, and then the computer and the software are started.
Parameter setting
1) Clicking a Method of a software Run interface, displaying edited Method information in a pull-down menu, selecting the Method, clicking New to create or selecting an existing operation Method in a list, and then editing by Edit.
2) The relevant parameters are set according to the length of the target segment to be interrupted by the sample. Specific parameters can be referred to as follows:
MicroTUBE AFA Fiber Screw-Cap-50ul sample-from 150bp to 800bp。
| 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 |
MicroTUBE AFA Fiber Snap-Cap-130ul sample-from 150bp to 1500bp。
| 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 |
running program
Clicking a 'Run' button on a Run interface to Run the program.
Shutdown software and machine
1) Emptying the water bath with a matched syringe; if the water Tray (Drip Tray) contains water, it is poured off and wiped dry with absorbent paper.
2) And (4) closing the software, then closing the instrument host, and finally closing the computer.
Step five: purification recovery and quality control of interrupted product
DNA fragments were selectively recovered using Yikang magnetic bead method DNA purification recovery reagent (Cat. No. YK003).
Note that: the selective recovery of DNA fragments is an optional step, and if the initial sample size is less than 50ng, the selective recovery of DNA fragments is not recommended, and the purification of DNA fragments can be directly carried out by referring to the instruction. In addition, when constructing DNA libraries of different sizes, the amount of magnetic beads selectively recovered from DNA fragments is different, and the specific amount of magnetic beads can be referred to as follows (if magnetic beads from manufacturers other than Yikang are used, the optimal amount of magnetic beads is required to be searched for).
1) Yield: not less than 25 mug before purification and not less than 12 mug after purification.
2) Fragment size: electrophoresis is carried out before purification, and more than 80% of the length of the product fragment is in the range of 200-300 bp.
Example 2
The method comprises the following steps: extraction of genomic DNA
Reference example 1 genomic DNA extraction method in step one.
Step two: disruption of Cot-1DNA
Referring to example 1, the method of disruption in step four, Cot-1DNA is disrupted to small fragments of about 30bp (actual disruption products are more than about 80% in the 25bp-35bp range).
Step three: MDA amplification reaction
And (3) performing MDA amplification by taking the genome DNA as a template and the broken Cot-1DNA as a primer. The specific operation steps refer to step three in example 1. Finally, the MDA amplification product is purified and quantified.
The column type purification is adopted, the product is recommended to be century CW2301M, and the specific operation is described in the specification. Quantification of MDA amplification products was performed using the Qubit.
Step four: fragmentation of MDA amplification product
Referring to the method interrupted in step four of example 1, the MDA amplification product was fragmented.
Step five: the purification, recovery and quality control of the product are interrupted.
DNA fragments were selectively recovered using Yikang magnetic bead method DNA purification recovery reagent (Cat. No. YK003). The recovered product was quantified using Qubit.
Example 3
This example demonstrates the effectiveness of the blocking reagents prepared by the two methods of the above examples:
preparation and sequencing of the target library:
DNA extraction, namely extracting the genome DNA of the FFPE sample by adopting a genome DNA extraction kit for fixed tissues in the Kangji century. The specific operation refers to the specification.
2. Library construction: the construction of the second generation sequencing Library is carried out by adopting the kang century NGS Fast DNA Library Prep Set for Illumina, and the specific operation is carried out by referring to an instruction book.
3. Hybridization, capture and elution of the library.
Taking 3 1ug of the same constructed library to a new centrifuge tube, sealing L1 with the sealing reagent prepared in example 1, sealing L2 with the sealing reagent prepared in example 2, sealing L3 with COT1DNA, and adding the rest components as follows:
| reagent | Dosage of |
| Libraries | 1ug |
| Repetitive sequence blocking reagent/COT 1DNA | 5ul |
| Universal blocking sequence | 1ul |
Concentrating with vacuum concentrator. For each concentrated library, the remaining components were added as follows.
| Reagent | Volume (ul) |
| 2X Hybridization Buffer | 8.5 |
| Hybridization Buffer Enhancer | 2.7 |
| Nuclease-free water | 1.8 |
After gentle vortex mixing, the mixture was left at room temperature for 5min, transferred to a new PCR tube, incubated on a PCR instrument at 95 ℃ for 10min (hot lid temperature 105 ℃) and taken out, 4ul of capture probe was immediately added, and incubated overnight at 50 ℃.
And (3) elution: elution was performed using the elution reagent and method for IDT.
And 4, sequencing the purified product NGS on a computer after PCR amplification.
The PCR system was prepared according to the following reagents:
| reagent | Volume of |
| Eluted DNA library | 30μL |
| PCR Buffer | 18μL |
| PCR Primer(25μM,for ILM) | 1μL |
| PCR Polymerase | 1μL |
| Total volume | 50ul |
And (3) preparing a PCR reaction system and putting the PCR reaction system into a PCR instrument for amplification, wherein the reaction procedure is as follows:
after the PCR reaction is finished, 1X Agencourt AMPure XP magnetic bead is adopted for purification, 25 mu l of nucleic-free water is adopted for elution (the concentration of the library is about 1-20 ng/mu l), 3 captured libraries are taken for Illumina on-machine sequencing, and the sequencing data volume is 200M. The data analysis results are as follows.
| Name of liberty | Data volume | Efficiency of capture | Effective depth of target area |
| L1 | 200M | 85％ | 1950X |
| L2 | 200M | 80％ | 2103X |
| L3 | 200M | 79％ | 2040X |
The effect of the blocking reagent prepared by the method of the patent can be close to the blocking efficiency of the commercial COT1DNA by the same sample and the same capture operation. Both preparation methods proved to be feasible.
The foregoing is directed to embodiments of the present invention, and it is understood that various modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention.
Sequence listing
<110> Yikang medical inspection Co., Ltd, Suzhou
<120> method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology
<130> GAI21CN4210
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 1
cctgaggtca ggagttcgag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 2
cccgagtagc tgggattaca 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 3
gggatctgca cccagaaaac 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 4
aaggaccagt ctctagcgga 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
gacaaatgct tcacacgggg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
gaaatggccg aggcgaaaac 20
Claims (10)
1. A method of preparing a blocking reagent for a repetitive sequence, the method comprising:
amplifying the genome DNA by adopting a multiple substitution amplification technology;
after purifying the amplification product, breaking the DNA fragment with the length of 200-300bp, and obtaining the product which is the blocking reagent of the repetitive sequence.
2. The method of claim 1, further comprising a process of extracting genomic DNA.
3. The method of claim 2, wherein the genomic DNA is extracted as follows:
when extracting genome DNA, adding nuclear lysis solution and protease K, SDS to fully crack tissues, digesting for 12-16 hours at 37-56 ℃; DNA was separated by centrifugation at 3000 and 10000rpm for 5-15 min.
4. The method of claim 1, wherein the genomic DNA is amplified using a multiple displacement amplification technique using the following primers:
primers of medium and/or high repeat sequence; or
The Cot-1DNA was broken and used as a random primer.
5. The method of claim 4, wherein the primers for moderately and/or highly repetitive sequences comprise one or more of Alu, Kpn, LINE families.
6. The method of claim 5, wherein the primers for moderately and/or highly repetitive sequences comprise at least one of each of the Alu, Kpn, LINE families.
7. The method of claim 4, wherein the Cot-1DNA is broken into small fragments of about 20-40bp when used as random primers.
8. The method of any one of claims 1 or 4 to 7, wherein the amplification of the genomic DNA using the multiple displacement amplification technique is performed by:
cracking the sample, neutralizing, centrifuging, adding the amplification mixed solution, and performing PCR amplification;
preferably, the temperature in the PCR instrument is set to be 30 ℃ for 8h during amplification; 5min at 65 ℃; the hot lid was 70 ℃.
9. The method of claim 8, wherein the lysing solution formulation used in the lysing comprises: 1.15. mu.l of lysis buffer, 0.35. mu.l of DTT and 1.5. mu.l of NF water per 1reaction volume;
the formula of the amplification mixed solution comprises: for each 1reaction volume, 37. mu.l of amplification buffer, 2. mu.l of DNA polymerase, 3. mu.l of primers, 0.5. mu.l of enhancer.
10. The method of claim 1, further comprising purifying and recovering the cleaved product and controlling the quality of the cleaved product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110778770.9A CN113444776A (en) | 2021-07-09 | 2021-07-09 | Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110778770.9A CN113444776A (en) | 2021-07-09 | 2021-07-09 | Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113444776A true CN113444776A (en) | 2021-09-28 |
Family
ID=77815646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110778770.9A Pending CN113444776A (en) | 2021-07-09 | 2021-07-09 | Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113444776A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957125A (en) * | 2021-10-21 | 2022-01-21 | 上海英基生物科技有限公司 | Cot DNA suitable for bisulfite sequencing, preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0511750A1 (en) * | 1991-04-09 | 1992-11-04 | The Regents Of The University Of California | Repeat sequence chromosome specific nucleic acid probes |
| US5427932A (en) * | 1991-04-09 | 1995-06-27 | Reagents Of The University Of California | Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using |
| US20030129626A1 (en) * | 2001-09-24 | 2003-07-10 | Nielsen Kirsten Vang | Methods, kits and compositions pertaining to the suppression of detectable probe binding to randomly distributed repeat sequences in genomic nucleic acid |
| CN108103052A (en) * | 2016-11-24 | 2018-06-01 | 安诺优达基因科技(北京)有限公司 | Improve the unicellular whole genome amplification and library constructing method of genome coverage |
| KR20180118454A (en) * | 2017-04-21 | 2018-10-31 | 주식회사 캔서롭 | Method for mass production of Cot-1 DNA |
| CN110409001A (en) * | 2019-07-25 | 2019-11-05 | 北京贝瑞和康生物技术有限公司 | The method and kit in a kind of building capture library |
-
2021
- 2021-07-09 CN CN202110778770.9A patent/CN113444776A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0511750A1 (en) * | 1991-04-09 | 1992-11-04 | The Regents Of The University Of California | Repeat sequence chromosome specific nucleic acid probes |
| US5427932A (en) * | 1991-04-09 | 1995-06-27 | Reagents Of The University Of California | Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using |
| US20030129626A1 (en) * | 2001-09-24 | 2003-07-10 | Nielsen Kirsten Vang | Methods, kits and compositions pertaining to the suppression of detectable probe binding to randomly distributed repeat sequences in genomic nucleic acid |
| US20180312912A1 (en) * | 2001-09-24 | 2018-11-01 | Applied Biosystems, Llc | Methods, Kits and Compositions Pertaining to the Suppression of the Detectable Probe Binding to Randomly Distributed Repeat Sequences Genomic Nucleic Acid |
| CN108103052A (en) * | 2016-11-24 | 2018-06-01 | 安诺优达基因科技(北京)有限公司 | Improve the unicellular whole genome amplification and library constructing method of genome coverage |
| KR20180118454A (en) * | 2017-04-21 | 2018-10-31 | 주식회사 캔서롭 | Method for mass production of Cot-1 DNA |
| CN110409001A (en) * | 2019-07-25 | 2019-11-05 | 北京贝瑞和康生物技术有限公司 | The method and kit in a kind of building capture library |
Non-Patent Citations (1)
| Title |
|---|
| 程东凯 等: "采用基因组测序技术进行胚胎种植前遗传学筛查的方法学研究及应用", 中华细胞与干细胞杂志(电子版), vol. 5, no. 02, pages 107 - 112 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957125A (en) * | 2021-10-21 | 2022-01-21 | 上海英基生物科技有限公司 | Cot DNA suitable for bisulfite sequencing, preparation method and application thereof |
| CN113957125B (en) * | 2021-10-21 | 2024-05-07 | 上海英基生物科技有限公司 | Cot DNA suitable for bisulfite sequencing, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3636769B1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| JP4304348B2 (en) | DNA isolation method | |
| EP1529841B1 (en) | RNA extraction method, RNA extraction reagent, and method for analyzing biological materials | |
| CN107955832B (en) | Primer set and method for simultaneously detecting thalassemia, deafness, phenylketonuria and hepatolenticular degeneration disease | |
| US20190352711A1 (en) | Method for Rapidly Constructing Amplicon Library Through One-Step Process | |
| WO2016050123A1 (en) | Pyrolysis composition as well as use and kit thereof, method for preparing nucleic acid by using pyrolysis composition, and method for nucleic acid analysis | |
| US6528256B1 (en) | Methods for identification and isolation of specific nucleotide sequences in cDNA and genomic DNA | |
| US20040126796A1 (en) | Extraction of DNA from biological samples | |
| CN113444776A (en) | Method for preparing blocking reagent of repetitive sequence by multiple substitution amplification technology | |
| WO2015159979A1 (en) | Method for recovering short-chain nucleic acids | |
| US10160965B2 (en) | Method and materials for nucleic acids extraction and purification | |
| CN115851876A (en) | Sequencing method for simultaneously obtaining whole genome transcription and protein-DNA binding information | |
| CN113215663B (en) | Construction method of gastric cancer targeted therapy genome library based on high-throughput sequencing and primers | |
| CN114836540A (en) | Kit for detecting BCR/-ABL1 fusion gene and use method thereof | |
| CN111235270B (en) | Tumor antigen expression detection primer and kit based on high-throughput sequencing method | |
| US10144951B2 (en) | Method and materials to deplete impurities for extraction and purification of nucleic acids from stool | |
| Lin et al. | Protocol for genomic DNA preparation from fresh or frozen serum for PCR amplification | |
| CN114164271B (en) | Primer pair, kit and method for detecting breast cancer susceptibility gene variation based on long fragment PCR | |
| CN111705111A (en) | Improved method for detecting gastric cancer frozen tissue open chromatin | |
| CN112080555A (en) | DNA methylation detection kit and detection method | |
| CN110777194A (en) | Denaturation-enhanced digital droplet PCR method for detecting highly fragmented samples | |
| CN112831555B (en) | Reference substance for detecting thalassemia gene and preparation method and application thereof | |
| CN114134206B (en) | FFPE sample RNA library and construction method thereof | |
| JP2014512190A (en) | Nucleic acid liquid phase extraction method and detection method thereof | |
| CN112795676B (en) | Kit for identifying visceral leishmaniasis infection pathogen insect species |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |