CN113004343A - Preparation method of second-generation sequencing capture probe - Google Patents
Preparation method of second-generation sequencing capture probe Download PDFInfo
- Publication number
- CN113004343A CN113004343A CN202110231889.4A CN202110231889A CN113004343A CN 113004343 A CN113004343 A CN 113004343A CN 202110231889 A CN202110231889 A CN 202110231889A CN 113004343 A CN113004343 A CN 113004343A
- Authority
- CN
- China
- Prior art keywords
- ammonolysis
- speed
- condition
- balancing
- rotating speed
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 47
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims description 11
- 238000000034 method Methods 0.000 claims abstract description 35
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 80
- 238000005915 ammonolysis reaction Methods 0.000 claims description 74
- 238000000746 purification Methods 0.000 claims description 47
- 229910052742 iron Inorganic materials 0.000 claims description 40
- 239000003153 chemical reaction reagent Substances 0.000 claims description 38
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 36
- 238000000703 high-speed centrifugation Methods 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 12
- 239000003755 preservative agent Substances 0.000 claims description 12
- 230000002335 preservative effect Effects 0.000 claims description 12
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 claims description 12
- 238000001819 mass spectrum Methods 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
- 230000003213 activating effect Effects 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 8
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000003517 fume Substances 0.000 claims description 4
- 238000010008 shearing Methods 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims 1
- 239000012452 mother liquor Substances 0.000 claims 1
- 239000007790 solid phase Substances 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 230000008878 coupling Effects 0.000 abstract description 3
- 238000010168 coupling process Methods 0.000 abstract description 3
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 125000004122 cyclic group Chemical group 0.000 abstract description 2
- 238000010511 deprotection reaction Methods 0.000 abstract description 2
- 238000009396 hybridization Methods 0.000 abstract description 2
- 239000000178 monomer Substances 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 150000008300 phosphoramidites Chemical class 0.000 abstract 1
- 239000000376 reactant Substances 0.000 abstract 1
- 238000003836 solid-state method Methods 0.000 abstract 1
- 238000007098 aminolysis reaction Methods 0.000 description 6
- 230000027832 depurination Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000010413 mother solution Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- RZEKVGVHFLEQIL-UHFFFAOYSA-N celecoxib Chemical compound C1=CC(C)=CC=C1C1=CC(C(F)(F)F)=NN1C1=CC=C(S(N)(=O)=O)C=C1 RZEKVGVHFLEQIL-UHFFFAOYSA-N 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- -1 phosphite amide triester Chemical class 0.000 description 1
- 238000007841 sequencing by ligation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for preparing a second-generation sequencing capture probe, which fixes DNA on a solid phase carrier by a solid phase phosphoramidite three-ester method, and gradually connects a plurality of nucleotide monomers by four steps of cyclic reaction of deprotection, coupling, capping and oxidation to finally complete the synthesis of a DNA chain, wherein the method has the advantages of high efficiency, quick coupling and stable initial reactant, the method for preparing the second-generation sequencing capture probe is a solid-state hybridization method, also called a solid-state method, which is essentially a chip which is fully distributed with probes, and is characterized in that the length of the probes is 50-105bp, the density and sequence of the probes can be considered to be set, relatively speaking, the higher the density of the probes in a section is, the higher the capture success rate is, and the problem that how to prepare the second-generation sequencing capture probe by using a solid-phase chemical method in the prior art is solved, provides a guarantee for the second-generation sequencing research.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a preparation method of a second-generation sequencing capture probe.
Background
Second generation DNA sequencing technology (abbreviation)"second generation sequencing"), also known as next generation sequencing technology, is the core of an epoch-making revolution in the first generation of sequencing technology. The existing technical platform mainly comprises Roche/454 GS FLX, Illumina/Sol-exa genome Analyzer, HeliScope of Helicos BioSciences companyTMSingle Mobile sequence, Polonator, available from Dana-her Motion, USA; and sequencing by ligation (i.e., positioning nucleic acid information by primers) with Applied Biosystems/SOLiD as a technical platformTMA system is provided. The sequencing principle applied by the technical platform is a circular microarray method.
The principle of hybrid capture is that probes are designed that can be partially or fully complementary to the target segment. The probe captures the target segment, the segment without designed probe is eluted and discarded, and then the probe and the capture segment are separated by denaturation, and the double-captured segment can be used for constructing a second-generation sequencing library.
The invention relates to a preparation method of a second generation sequencing capture probe, which is a solid-state hybridization method essentially, also called a solid-phase method, and is essentially a chip, wherein the chip is fully distributed with probes, the preparation method is characterized in that DNA probes are different in length of 50-105bp, the density and the sequence of the probes can be considered as setting, and relatively speaking, the higher the density of the probes for one section is, the higher the capture success rate is.
Disclosure of Invention
The invention aims to provide a preparation method of a second-generation sequencing capture probe, and solves the problem that how to prepare the second-generation sequencing capture probe by using a solid phase chemical method in the prior art provides guarantee for the second-generation sequencing research.
The purpose of the invention can be realized by the following technical scheme:
a preparation method of a second generation sequencing capture probe comprises the following steps:
the method comprises the following steps: ammonolysis treatment;
step two: activating the purification column;
step three: purifying;
step four: and (4) measuring and detecting mass spectra.
As a further scheme of the invention: the specific process of the ammonolysis treatment is as follows:
s21, pretreatment of ammonolysis: adding 50 mu L of diethylamine with the mass fraction of 10% into each hole of the synthetic plate, standing for 10min, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then adding 50 mu L of diethylamine with the volume fraction of 10% into each hole, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then washing for 2 times by using acetonitrile, and carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min after the 2 nd washing;
s22, ammonolysis:
s221, first-step ammonolysis: taking a new 384-plate to be adhered with a label, wherein the label data is the same as that of the original plate, placing the new 384-plate on the bottom layer of the synthetic plate, adding 50 mu L of ice ammonia water into each hole of the synthetic plate, wrapping the synthetic plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the synthetic plate in an iron aminolysis box, and then placing the iron aminolysis box in an oven with the temperature of 45 ℃ for ammonolysis for 1 h;
s222: after the ammonolysis is finished, taking out and cooling, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and placing in a fume hood, shearing off the preservative film by using scissors, adding 50 mu L of ice ammonia water into each hole of the synthetic plate again, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and keeping in a collecting plate;
s223: the second step of ammonolysis: covering a plastic cover on the collecting plate, wrapping the collecting plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the collecting plate in an ammonolysis iron box, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for ammonolysis for 3 hours;
s224: and after the ammonolysis is finished, taking out the ammonolysis iron box, cooling the ammonolysis iron box in a cool and ventilated place for 15-20min, cooling the ammonolysis iron box, centrifuging the ammonolysis iron box at a high speed for 1min under the condition that the rotating speed is 4000r/min, opening the ammonolysis iron box, removing a plastic cover, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for drying residual ammonia water.
As a further scheme of the invention: the specific process of activating the purification column is as follows:
s31: adding acetonitrile with the volume fraction of 100% in the corresponding volume into each hole of the collecting plate, standing for 20s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s32: repeating the step S31 for 3 times;
s33: then adding a corresponding volume of 0.1mol/L TEAA solution into each hole of the collecting plate for cleaning, standing for 20s, and then balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s34: the step of S33 was repeated 3 more times to obtain an activated purification column.
As a further scheme of the invention: the specific process of the purification is as follows:
s41: adding 100 mu L of M1-M2 mixed solution into each hole of the collection plate, standing for 2min until the sample is fully contacted with the solution, and obtaining a sample to be purified;
s42, transferring the sample, adsorbing and passing through the column: transferring a sample to be purified into an activated purification column, standing for 5s, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 100r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 300r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 800r/min, then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 1600r/min, and then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 2400 r/min;
s43: repeating the step S42 for 4-6 times to ensure that all the components are adsorbed to the purification column, storing the mother solution, and continuing the purification column in the following steps;
s44: adding 100 mu L of 0.1mol/L TEAA solution into the purification column, cleaning, standing for 5s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2400 r/min;
s45: repeating the step S44 for 2 times;
s46: placing a 96-hole collecting plate at the bottom of the purification column, adding 100 μ L of M4 reagent into the purification column, standing, balancing at 2400r/min, centrifuging at high speed for 1min, and observing the color of the liquid in the collecting plate; under normal conditions, the color is orange red, and the redder the color is, the more DNA fragments adsorbed are, but the longer the time is, the better the time is, and depurination can occur after the time is too long;
s47: placing a new 384-hole collection plate at the bottom of the purification column, adding 100 μ L of M5 reagent into the purification column, standing for 30s, balancing and high-speed centrifuging at a rotation speed of 300r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 800r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 1600r/min for 1min, and balancing and high-speed centrifuging at a rotation speed of 2400r/min for 1 min.
As a further scheme of the invention: the specific processes of the measurement and the mass spectrum detection are as follows:
s51: placing a 384-hole collection plate on a shaking instrument, shaking and uniformly mixing for 30min, taking 2 mu L of primers, uniformly mixing with 48 mu L of water, and placing the mixture on a microplate reader for measurement;
s52: 5 mu L of the primer is uniformly mixed with 60 mu L of water and then the mass spectrum detection is carried out.
As a further scheme of the invention: the TEAA solution is a mixture of triethylamine, acetic acid and water according to a volume ratio of 14:6: 80.
As a further scheme of the invention: in the step S41, the mixed solution of M1-M2 is a mixture of M1 reagent and M2 reagent in a volume ratio of 2:3, wherein the M1 reagent is a triethylamine solution with a volume fraction of 3%, and the M2 reagent is a mixture of N, N-dimethylformamide, triethylamine and water in a volume ratio of 5:2: 93; the M4 reagent in step S46 is trifluoroacetic acid with a volume fraction of 2%; the M5 reagent in step S47 is 25% volume fraction acetonitrile.
As a further scheme of the invention: the 768 synthesizer is used in the synthesis process, the 768 synthesizer is operated, at least the first base complete cycle is monitored, the reagent spraying, the carrier integrity, the reagent permeation and the carrier reagent draining conditions are monitored emphatically, the DMT color when the first base is finished is observed emphatically, the operation state is observed irregularly in the midway, and whether the reagent amount is sufficient or not and whether the reagent liquid level of the buffer bottle is over the bottom of the reagent pipeline or not need to be checked.
The invention has the beneficial effects that:
the invention relates to a preparation method of a second-generation sequencing capture probe, which is characterized in that DNA is fixed on a solid-phase carrier by a solid-phase phosphite amide triester method, and a plurality of nucleotide monomers are gradually connected in sequence through four steps of cyclic reaction of deprotection, coupling, capping and oxidation, so that the synthesis of a DNA chain is finally completed.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the embodiment is a preparation method of a second generation sequencing capture probe, which comprises the following steps:
the method comprises the following steps: ammonolysis treatment;
step two: activating the purification column;
step three: purifying;
step four: and (4) measuring and detecting mass spectra.
The specific process of the ammonolysis treatment is as follows:
s21, pretreatment of ammonolysis: adding 50 mu L of diethylamine with the mass fraction of 10% into each hole of the synthetic plate, standing for 10min, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then adding 50 mu L of diethylamine with the volume fraction of 10% into each hole, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then washing for 2 times by using acetonitrile, and carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min after the 2 nd washing;
s22, ammonolysis:
s221, first-step ammonolysis: taking a new 384-plate to be adhered with a label, wherein the label data is the same as that of the original plate, placing the new 384-plate on the bottom layer of the synthetic plate, adding 50 mu L of ice ammonia water into each hole of the synthetic plate, wrapping the synthetic plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the synthetic plate in an iron aminolysis box, and then placing the iron aminolysis box in an oven with the temperature of 45 ℃ for ammonolysis for 1 h;
s222: after the ammonolysis is finished, taking out and cooling, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and placing in a fume hood, shearing off the preservative film by using scissors, adding 50 mu L of ice ammonia water into each hole of the synthetic plate again, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and keeping in a collecting plate;
s223: the second step of ammonolysis: covering a plastic cover on the collecting plate, wrapping the collecting plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the collecting plate in an ammonolysis iron box, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for ammonolysis for 3 hours;
s224: and after the ammonolysis is finished, taking out the ammonolysis iron box, cooling the ammonolysis iron box in a cool and ventilated place for 15min, cooling the ammonolysis iron box, centrifuging the ammonolysis iron box at a high speed for 1min under the condition that the rotating speed is 4000r/min, opening the ammonolysis iron box, removing a plastic cover, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for drying residual ammonia water.
The specific process of activating the purification column is as follows:
s31: adding acetonitrile with the volume fraction of 100% in the corresponding volume into each hole of the collecting plate, standing for 20s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s32: repeating the step S31 for 3 times;
s33: then adding a corresponding volume of 0.1mol/L TEAA solution into each hole of the collecting plate for cleaning, standing for 20s, and then balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s34: the step of S33 was repeated 3 more times to obtain an activated purification column.
The specific process of the purification is as follows:
s41: adding 100 mu L of M1-M2 mixed solution into each hole of the collection plate, standing for 2min until the sample is fully contacted with the solution, and obtaining a sample to be purified;
s42, transferring the sample, adsorbing and passing through the column: transferring a sample to be purified into an activated purification column, standing for 5s, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 100r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 300r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 800r/min, then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 1600r/min, and then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 2400 r/min;
s43: repeating the step S42 for 4 times to ensure that all the components are adsorbed to the purification column, preserving the mother solution, and continuing the purification column in the following steps;
s44: adding 100 mu L of 0.1mol/L TEAA solution into the purification column, cleaning, standing for 5s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2400 r/min;
s45: repeating the step S44 for 2 times;
s46: placing a 96-hole collecting plate at the bottom of the purification column, adding 100 μ L of M4 reagent into the purification column, standing, balancing at 2400r/min, centrifuging at high speed for 1min, and observing the color of the liquid in the collecting plate; under normal conditions, the color is orange red, and the redder the color is, the more DNA fragments adsorbed are, but the longer the time is, the better the time is, and depurination can occur after the time is too long;
s47: placing a new 384-hole collection plate at the bottom of the purification column, adding 100 μ L of M5 reagent into the purification column, standing for 30s, balancing and high-speed centrifuging at a rotation speed of 300r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 800r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 1600r/min for 1min, and balancing and high-speed centrifuging at a rotation speed of 2400r/min for 1 min.
The specific processes of the measurement and the mass spectrum detection are as follows:
s51: placing a 384-hole collection plate on a shaking instrument, shaking and uniformly mixing for 30min, taking 2 mu L of primers, uniformly mixing with 48 mu L of water, and placing the mixture on a microplate reader for measurement;
s52: 5 mu L of the primer is uniformly mixed with 60 mu L of water and then the mass spectrum detection is carried out.
The TEAA solution is a mixture of triethylamine, acetic acid and water according to a volume ratio of 14:6: 80.
In the step S41, the mixed solution of M1-M2 is a mixture of M1 reagent and M2 reagent in a volume ratio of 2:3, wherein the M1 reagent is a triethylamine solution with a volume fraction of 3%, and the M2 reagent is a mixture of N, N-dimethylformamide, triethylamine and water in a volume ratio of 5:2: 93; the M4 reagent in step S46 is trifluoroacetic acid with a volume fraction of 2%; the M5 reagent in step S47 is 25% volume fraction acetonitrile.
Example 2:
the embodiment is a preparation method of a second generation sequencing capture probe, which comprises the following steps:
the method comprises the following steps: ammonolysis treatment;
step two: activating the purification column;
step three: purifying;
step four: and (4) measuring and detecting mass spectra.
The specific process of the ammonolysis treatment is as follows:
s21, pretreatment of ammonolysis: adding 50 mu L of diethylamine with the mass fraction of 10% into each hole of the synthetic plate, standing for 10min, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then adding 50 mu L of diethylamine with the volume fraction of 10% into each hole, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then washing for 2 times by using acetonitrile, and carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min after the 2 nd washing;
s22, ammonolysis:
s221, first-step ammonolysis: taking a new 384-plate to be adhered with a label, wherein the label data is the same as that of the original plate, placing the new 384-plate on the bottom layer of the synthetic plate, adding 50 mu L of ice ammonia water into each hole of the synthetic plate, wrapping the synthetic plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the synthetic plate in an iron aminolysis box, and then placing the iron aminolysis box in an oven with the temperature of 45 ℃ for ammonolysis for 1 h;
s222: after the ammonolysis is finished, taking out and cooling, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and placing in a fume hood, shearing off the preservative film by using scissors, adding 50 mu L of ice ammonia water into each hole of the synthetic plate again, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and keeping in a collecting plate;
s223: the second step of ammonolysis: covering a plastic cover on the collecting plate, wrapping the collecting plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the collecting plate in an ammonolysis iron box, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for ammonolysis for 3 hours;
s224: and after the ammonolysis is finished, taking out the ammonolysis iron box, cooling the ammonolysis iron box in a cool and ventilated place for 20min, cooling the ammonolysis iron box, centrifuging the ammonolysis iron box at a high speed for 1min under the condition that the rotating speed is 4000r/min, opening the ammonolysis iron box, removing a plastic cover, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for drying residual ammonia water.
The specific process of activating the purification column is as follows:
s31: adding acetonitrile with the volume fraction of 100% in the corresponding volume into each hole of the collecting plate, standing for 20s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s32: repeating the step S31 for 3 times;
s33: then adding a corresponding volume of 0.1mol/L TEAA solution into each hole of the collecting plate for cleaning, standing for 20s, and then balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s34: the step of S33 was repeated 3 more times to obtain an activated purification column.
The specific process of the purification is as follows:
s41: adding 100 mu L of M1-M2 mixed solution into each hole of the collection plate, standing for 2min until the sample is fully contacted with the solution, and obtaining a sample to be purified;
s42, transferring the sample, adsorbing and passing through the column: transferring a sample to be purified into an activated purification column, standing for 5s, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 100r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 300r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 800r/min, then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 1600r/min, and then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 2400 r/min;
s43: repeating the step S42 for 6 times to ensure that all the components are adsorbed to the purification column, preserving the mother solution, and continuing the purification column in the following steps;
s44: adding 100 mu L of 0.1mol/L TEAA solution into the purification column, cleaning, standing for 5s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2400 r/min;
s45: repeating the step S44 for 2 times;
s46: placing a 96-hole collecting plate at the bottom of the purification column, adding 100 μ L of M4 reagent into the purification column, standing, balancing at 2400r/min, centrifuging at high speed for 1min, and observing the color of the liquid in the collecting plate; under normal conditions, the color is orange red, and the redder the color is, the more DNA fragments adsorbed are, but the longer the time is, the better the time is, and depurination can occur after the time is too long;
s47: placing a new 384-hole collection plate at the bottom of the purification column, adding 100 μ L of M5 reagent into the purification column, standing for 30s, balancing and high-speed centrifuging at a rotation speed of 300r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 800r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 1600r/min for 1min, and balancing and high-speed centrifuging at a rotation speed of 2400r/min for 1 min.
The specific processes of the measurement and the mass spectrum detection are as follows:
s51: placing a 384-hole collection plate on a shaking instrument, shaking and uniformly mixing for 30min, taking 2 mu L of primers, uniformly mixing with 48 mu L of water, and placing the mixture on a microplate reader for measurement;
s52: 5 mu L of the primer is uniformly mixed with 60 mu L of water and then the mass spectrum detection is carried out.
The TEAA solution is a mixture of triethylamine, acetic acid and water according to a volume ratio of 14:6: 80.
In the step S41, the mixed solution of M1-M2 is a mixture of M1 reagent and M2 reagent in a volume ratio of 2:3, wherein the M1 reagent is a triethylamine solution with a volume fraction of 3%, and the M2 reagent is a mixture of N, N-dimethylformamide, triethylamine and water in a volume ratio of 5:2: 93; the M4 reagent in step S46 is trifluoroacetic acid with a volume fraction of 2%; the M5 reagent in step S47 is 25% volume fraction acetonitrile.
The mass spectrometric detection of examples 1-2 is shown in the following table:
| sample (I) | Mass spectrometric detection results |
| Example 1 | Has correct molecular weight and purity of 82.3% |
| Example 2 | Has correct molecular weight and purity of 83.8% |
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.
Claims (7)
1. The preparation method of the second-generation sequencing capture probe is characterized by comprising the following steps of:
the method comprises the following steps: ammonolysis treatment;
step two: activating the purification column;
step three: purifying;
step four: and (4) measuring and detecting mass spectra.
2. The method for preparing a secondary sequencing capture probe according to claim 1, wherein the ammonolysis treatment comprises the following steps:
s21, pretreatment of ammonolysis: adding 50 mu L of diethylamine with the mass fraction of 10% into each hole of the synthetic plate, standing for 10min, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then adding 50 mu L of diethylamine with the volume fraction of 10% into each hole, carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min, then washing for 2 times by using acetonitrile, and carrying out high-speed centrifugation for 1min under the condition of the rotating speed of 4000r/min after the 2 nd washing;
s22, ammonolysis:
s221, first-step ammonolysis: taking a new 384-plate to be labeled, placing the new 384-plate on the bottom layer of a synthetic plate, adding 50 mu L of ice ammonia water into each hole of the synthetic plate, wrapping the new 384-plate with 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the new 384-plate in an ammonolysis iron box, and placing the ammonolysis iron box in an oven at the temperature of 45 ℃ for ammonolysis for 1 hour;
s222: after the ammonolysis is finished, taking out and cooling, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and placing in a fume hood, shearing off the preservative film by using scissors, adding 50 mu L of ice ammonia water into each hole of the synthetic plate again, centrifuging at a high speed for 1min under the condition that the rotating speed is 4000r/min, taking out and keeping in a collecting plate;
s223: the second step of ammonolysis: covering a plastic cover on the collecting plate, wrapping the collecting plate by using 3 transverse layers and 3 vertical layers of preservative films to ensure tightness and no air leakage, placing the collecting plate in an ammonolysis iron box, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for ammonolysis for 3 hours;
s224: and after the ammonolysis is finished, taking out the ammonolysis iron box, cooling the ammonolysis iron box in a cool and ventilated place for 15-20min, cooling the ammonolysis iron box, centrifuging the ammonolysis iron box at a high speed for 1min under the condition that the rotating speed is 4000r/min, opening the ammonolysis iron box, removing a plastic cover, and then placing the ammonolysis iron box in an oven at the temperature of 90 ℃ for drying residual ammonia water.
3. The method for preparing a secondary sequencing capture probe according to claim 1, wherein the specific process of activating the purification column is as follows:
s31: adding acetonitrile with the volume fraction of 100% in the corresponding volume into each hole of the collecting plate, standing for 20s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s32: repeating the step S31 for 3 times;
s33: then adding a corresponding volume of 0.1mol/L TEAA solution into each hole of the collecting plate for cleaning, standing for 20s, and then balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2800 r/min;
s34: the step of S33 was repeated 3 more times to obtain an activated purification column.
4. The method for preparing a secondary sequencing capture probe according to claim 1, wherein the purification comprises the following steps:
s41: adding 100 mu L of M1-M2 mixed solution into each hole of the collection plate, and standing for 2min to obtain a sample to be purified;
s42, transferring the sample, adsorbing and passing through the column: transferring a sample to be purified into an activated purification column, standing for 5s, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 100r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 300r/min, then carrying out balancing high-speed centrifugation for 5min under the condition that the rotating speed is 800r/min, then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 1600r/min, and then carrying out balancing high-speed centrifugation for 2min under the condition that the rotating speed is 2400 r/min;
s43: repeating the step S42 for 4-6 times, storing the mother liquor, and continuing the next step by using the purification column;
s44: adding 100 mu L of 0.1mol/L TEAA solution into the purification column, cleaning, standing for 5s, and balancing and centrifuging at high speed for 1min under the condition that the rotating speed is 2400 r/min;
s45: repeating the step S44 for 2 times;
s46: placing a 96-hole collecting plate at the bottom of the purification column, adding 100 μ L of M4 reagent into the purification column, standing, balancing at 2400r/min, centrifuging at high speed for 1min, and observing the color of the liquid in the collecting plate;
s47: placing a new 384-hole collection plate at the bottom of the purification column, adding 100 μ L of M5 reagent into the purification column, standing for 30s, balancing and high-speed centrifuging at a rotation speed of 300r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 800r/min for 1min, balancing and high-speed centrifuging at a rotation speed of 1600r/min for 1min, and balancing and high-speed centrifuging at a rotation speed of 2400r/min for 1 min.
5. The method for preparing a secondary sequencing capture probe according to claim 1, wherein the specific processes of measurement and mass spectrometry detection are as follows:
s51: placing a 384-hole collection plate on a shaking instrument, shaking and uniformly mixing for 30min, taking 2 mu L of primers, uniformly mixing with 48 mu L of water, and placing the mixture on a microplate reader for measurement;
s52: 5 mu L of the primer is uniformly mixed with 60 mu L of water and then the mass spectrum detection is carried out.
6. The method for preparing a secondary sequencing capture probe according to claim 3, wherein the TEAA solution is a mixture of triethylamine, acetic acid and water in a volume ratio of 14:6: 80.
7. The method for preparing a secondary sequencing capture probe according to claim 4, wherein the mixture of M1-M2 in step S41 is a mixture of M1 reagent and M2 reagent in a volume ratio of 2:3, wherein M1 reagent is a triethylamine solution with a volume fraction of 3%, and M2 reagent is a mixture of N, N-dimethylformamide, triethylamine and water in a volume ratio of 5:2: 93; the M4 reagent in step S46 is trifluoroacetic acid with a volume fraction of 2%; the M5 reagent in step S47 is 25% volume fraction acetonitrile.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110231889.4A CN113004343A (en) | 2021-03-02 | 2021-03-02 | Preparation method of second-generation sequencing capture probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110231889.4A CN113004343A (en) | 2021-03-02 | 2021-03-02 | Preparation method of second-generation sequencing capture probe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113004343A true CN113004343A (en) | 2021-06-22 |
Family
ID=76402797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110231889.4A Pending CN113004343A (en) | 2021-03-02 | 2021-03-02 | Preparation method of second-generation sequencing capture probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113004343A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108395462A (en) * | 2017-12-13 | 2018-08-14 | 通用生物系统（安徽）有限公司 | A kind of novel Y-STR locus detection DNA probe synthetic method |
| CN109134541A (en) * | 2018-09-10 | 2019-01-04 | 伯科生物医学科技（北京）有限公司 | Long-chain biological element marker and its preparation method and application |
| CN111704644A (en) * | 2020-08-18 | 2020-09-25 | 苏州金唯智生物科技有限公司 | Ammonolysis solution and ammonolysis method |
| CN111710362A (en) * | 2020-08-20 | 2020-09-25 | 上海思路迪医学检验所有限公司 | Design method and application of capture probe based on next generation sequencing |
-
2021
- 2021-03-02 CN CN202110231889.4A patent/CN113004343A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108395462A (en) * | 2017-12-13 | 2018-08-14 | 通用生物系统（安徽）有限公司 | A kind of novel Y-STR locus detection DNA probe synthetic method |
| CN109134541A (en) * | 2018-09-10 | 2019-01-04 | 伯科生物医学科技（北京）有限公司 | Long-chain biological element marker and its preparation method and application |
| CN111704644A (en) * | 2020-08-18 | 2020-09-25 | 苏州金唯智生物科技有限公司 | Ammonolysis solution and ammonolysis method |
| CN111710362A (en) * | 2020-08-20 | 2020-09-25 | 上海思路迪医学检验所有限公司 | Design method and application of capture probe based on next generation sequencing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109811045B (en) | Construction method and application of high-throughput single-cell full-length transcriptome sequencing library | |
| CN106754343A (en) | DNA sequencing apparatus and system based on pyrosequencing | |
| CN106755292A (en) | A kind of phosphoric acid modifies the nucleic acid molecule sequence measurement of fluorogen | |
| RU96103646A (en) | REAGENT-MARKER AND METHOD FOR CARRYING OUT ANALYSIS | |
| CN105002569A (en) | Transcriptome library and construction method thereof | |
| CN111321208A (en) | Database building method based on high-throughput sequencing | |
| CN105925562A (en) | Method and kit for enriching 4000 human pathogenic target genes | |
| CN117051481B (en) | Preparation method and application of space bar code chip | |
| CN106399533B (en) | A kind of method and composition removing ribosomes nucleic acid rRNA in total serum IgE sample | |
| CN113004343A (en) | Preparation method of second-generation sequencing capture probe | |
| CN110042148B (en) | Method for efficiently acquiring chloroplast DNA sequencing data and application thereof | |
| CN106755506A (en) | Kit for detecting genetic mutation in tumour FFPE samples | |
| CN111471746A (en) | NGS library preparation joint for detecting low mutation abundance sample and preparation method thereof | |
| CN102140509B (en) | Gene mutation detection method based on nucleic acid amplification on solid carrier | |
| CN112979737A (en) | Method for reducing fluorescence background of Taqman probe | |
| CN108611350A (en) | 16S rDNA microorganism fungus kinds identification primer system, kit and application | |
| CN111733459A (en) | DNA coding compound library and screening method thereof | |
| CN106929564B (en) | Breast cancer susceptibility gene detection kit | |
| CN113550013B (en) | Method for rapidly constructing RRBS sequencing library by using formalin-fixed paraffin embedded sample | |
| CN106754309B (en) | The full-automatic library RNA preparation facilities applied to second generation high-flux sequence | |
| CN111197076A (en) | Antihypertensive drug irbesartan medication guidance and gene detection kit | |
| CN115478114A (en) | Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection | |
| CN108037257B (en) | test method and device for researching influence of iron ions on anaerobic ammonia oxidation energy efficiency | |
| CN106591486A (en) | Kit for detecting blood disease related gene variation | |
| CN112877442A (en) | 212Y-SNP forensic medicine composite amplification kit based on MALDI-TOF MS platform detection |
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 | ||
| CB02 | Change of applicant information |
Address after: 239000 No. 69, qifushi West Road, Chuzhou economic and Technological Development Zone, Anhui Province Applicant after: General Biology (Anhui) Co.,Ltd. Address before: 239000 No. 69, qifushi West Road, Chuzhou economic and Technological Development Zone, Anhui Province Applicant before: GENERAL BIOSYSTEMS (ANHUI), Inc. |
|
| CB02 | Change of applicant information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210622 |
|
| RJ01 | Rejection of invention patent application after publication |