CN106701993A - Trace nucleic acid sequencing pretreatment method based on magnetic bead coating and kit - Google Patents
Trace nucleic acid sequencing pretreatment method based on magnetic bead coating and kit Download PDFInfo
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- 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
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Abstract
The invention discloses a trace nucleic acid sequencing pretreatment method based on magnetic bead coating and a kit. The trace nucleic acid sequencing pretreatment method based on magnetic bead coating comprises the following steps of: firstly coating DNA (deoxyribonucleic acid) to be treated with magnetic beads, carrying out tail end repairing on the DNA to be treated, which is coated with the magnetic beads, purifying, leaving the magnetic beads, carrying out A addition reaction, purifying again, leaving the magnetic beads, carrying out joining reaction, purifying, discarding the magnetic beads, carrying out PCR (polymerase chain reaction), and purifying by adding the magnetic beads after completing polymerase chain reaction to obtain the treated DNA. The trace nucleic acid sequencing pretreatment method based on magnetic bead coating has the beneficial effects that the frequency of transferring trace nucleic acids in a sequencing pretreatment process is effectively reduced by coating the DNA with the magnetic beads, thereby greatly reducing the loss of the nucleic acids in a sequencing pretreatment process; reaction conditions are mild; meanwhile, the DNA damage is also reduced; the success rate of trace nucleic acid sequencing pretreatment is increased to 90% or above; the method has the characteristics of high success rate, high quality and low initial amount in comparison with a traditional nucleic acid sequencing pretreatment method; and used reagent raw materials are low in cost, simple and easily available.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to for DNA technique field, one is particularly related to
Plant based on magnetic bead coated trace dna sequencing pre-treating method and kit.
Background technology
DNA is the material base of human inheritance, in order to understand fully functions and knot of the DNA in human inheritance and growth course
Structure, the detection of DNA sequence dna becomes the most important means of researching DNA.Sanger has been invented with milestone significance within 1977
End terminates PCR sequencing PCR, and the same year, A.M.Maxam and W.Gilbert invented chemical degradation method.Sanger methods because it is not only easy but also
Quickly, and by follow-up continuous improvement, the main flow of first generation DNA sequencing is become.With the continuous innovation of science and technology, the
Generation DNA sequencing technology (Sanger) cannot meet the demand of sequencing, and nineteen ninety, United States Non-Provisional starts the cross-centennial " mankind
Genome plan ", plan lasts 15 years, furnishes funds for 3,000,000,000 dollars, decodes mankind itself's heredity secret, and the mankind have stepped into Post genome
Epoch.With the massive demand to DNA information, the sequencing of two generations is born, and the technology is based on a sequencing technologies, generation in synthesis
There are Roche (454), Illumina (Solexa), ABI (SOLID) in table company.
Two generation sequenators cannot directly read DNA sequence dna in itself could be placed, it is necessary to DNA first to be carried out homogenization pre-treatment
Recognized by it in sequenator.Therefore, DNA sequencing pre-treatment becomes the key technology of two generation sequencing technologies.Classical core
Sour pre-treatment step is to be repaired by end, add A to react, connect sequence measuring joints, PCR to complete, will be by 4 between this 4 steps
Secondary purification step, because purification step is various, can cause DNA originating demand amounts big, it usually needs more than 1ug.
As two generation sequencing technologies deepen continuously medical domain, increasing clinical sample is used for the sequencing of two generations, so
And clinical sample is very precious, amount of DNA is rare, DNA total amounts needed for the nucleic acid sequencing pre-treatment of classics is extremely difficult to, so as to turn into
The technology is applied to the bottleneck problem of clinical sample.
Therefore, how reducing DNA original amounts and improving trace dna sample sequencing pre-treatment success rate becomes industry
Current primary problem to be solved.
The content of the invention
The main object of the present invention is aiming above present situation, there is provided before one kind is based on the coated trace dna sequencing of magnetic bead
Processing method and kit, to overcome deficiency of the prior art.
To realize aforementioned invention purpose, the technical solution adopted by the present invention includes：
The embodiment of the invention provides a kind of coated trace dna of magnetic bead that is based on and pre-treating method is sequenced, it includes：
Pending DNA is first carried out into magnetic bead coating, the coated pending DNA of magnetic bead end reparation is carried out into thereafter, then
Purifying, leaves magnetic bead, carries out plus A reactions, and repurity afterwards leaves magnetic bead, is attached reaction, afterwards repurity, abandons magnetic
Pearl, enters performing PCR reaction, after the completion of PCR reactions, adds magnetic bead and is purified, the DNA after being processed.
Among some embodiments, when carrying out magnetic bead coating, the pending DNA is with the volumetric usage ratio of magnetic bead
0.5~1.2:1.5~2.0, further, the magnetic bead is AmpureXP magnetic beads.
Among some embodiments, the pre-treating method also includes：To adding in the coated pending DNA of magnetic bead
To carry out end reparation, first reagent is included one reagent：10X T4 DNA connection buffer solution, bovine serum albumin, ATP,
DNTPs, T4 phosphorylating kinase, T4 archaeal dna polymerases, Klenow Large fragment polymerases and ultra-pure water.
Further, the temperature program of the end reparation is：5min~20min is incubated at prior to 10 DEG C~15 DEG C, it
After 5min~20min is incubated at 20 DEG C~30 DEG C, most after 4 DEG C of preservations.
Further, the pH of first reagent is 7.3~8.5, and first reagent is included：10X T4 DNA connections are slow
The parts by volume of fliud flushing 2~20, the parts by volume of 1mg/mL bovine serum albumen solutions 2~10, the parts by volume of 10mM ATP solution 2~10,10mM
The parts by volume of dNTPs solution 1~5, the parts by volume of T4 phosphorylating kinases solution 2~10, the parts by volume of T4 archaeal dna polymerases solution 2~10,
The parts by volume of Klenow Large fragment polymerases solution 0.5~1.5, the parts by volume of ultra-pure water 10~50.
Among some embodiments, the pre-treating method is additionally included in be carried out plus A reactions are preceding adds the 3rd reagent, institute
The 3rd reagent is stated to include：10 × Klenow polymerizations buffer solution, adenine, Klenow polymerases and ultra-pure water.
Specifically, the temperature program of described plus A reactions is：At prior to 30 DEG C~40 DEG C be incubated 5min~60min, after
4 DEG C of preservations；
Further, the pH of the 3rd reagent is 7.3~8.5, and the 3rd reagent is included：10 × Klenow polymerizations are slow
The parts by volume of fliud flushing 2~10, the parts by volume of 1mM adenine solutions 2~20, the parts by volume of exo-Klenow polymerase solutions 2~10 and super
The parts by volume of pure water 20~50.
Among some embodiments, the pre-treating method is additionally included in before being attached reaction and adds the 4th reagent,
4th reagent is included：2 × DNA connections buffer solution, DNA ligase, Illumina sequence measuring joints and ultra-pure water.
Preferably, the temperature program of the coupled reaction is：5min~20min is incubated at prior to 20 DEG C~30 DEG C, afterwards
In 4 DEG C of preservations.
Further, the pH of the 4th reagent is 7.3~8.5, and the 4th reagent is included：2 × DNA connects buffer solution
5~25 parts by volume, the parts by volume of DNA ligase solution 1~5, the parts by volume of Illumina sequence measuring joints solution 0.5~3 and ultra-pure water
10~50 parts by volume.
Among some embodiments, the pre-treating method also includes：After abandoning magnetic bead, add before entering performing PCR reaction
Entering the 5th reagent carries out DNA wash-outs, is reacted to adding the 6th reagent to enter performing PCR in the DNA after wash-out；
Wherein, the 5th reagent is included：Trishydroxymethylaminomethane, HCl and ultra-pure water, the 6th reagent are included：2
× KAPA exo+ polymerases premixed liquid and Illumina sequencing primer mixed liquors.
It is more highly preferred to, the temperature program of the PCR reactions is：At a temperature of prior to 90 DEG C~100 DEG C preheat 1min~
5min, then carries out 2~20 circulations, and each circulation includes：Successively at 90 DEG C~100 DEG C be incubated 10s~50s, 55 DEG C~
At 72 DEG C be incubated 10s~50s, at 65 DEG C~85 DEG C be incubated 10s~50s, thereafter, at 60 DEG C~80 DEG C be incubated 1min~
10min, 4 DEG C of preservations.
Further, the pH of the 5th reagent is 7.3~8.5, and the consumption of the 5th reagent is 6uL~20uL, three
The concentration of hydroxymethyl aminomethane is 5mM-20mM；
And/or, the pH of the 6th reagent is 7.3~8.5, and the 6th reagent is included：2 × KAPA HiFi high-fidelities
The parts by volume of polymerase premixed liquid 5~25, the parts by volume of Illumina sequencing primers mixed liquor 1~5.
Among some embodiments, the second reagent is added in the purification step, the pH of second reagent is 7.3
~8.5, second reagent is included：10~30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water.
It is more highly preferred to, the pH of second reagent is 8.0, and second reagent is included：20wt% PEG 8000s,
2.5M sodium chloride and ultra-pure water.
The embodiment of the present invention additionally provides a kind of trace dna sequencing pretreatment reagent kit, and it is included：
First reagent, including 10X T4 DNA connection buffer solution, bovine serum albumin, ATP, dNTPs, T4 phosphorylating kinase,
T4 archaeal dna polymerases, Klenow Large fragment polymerases and ultra-pure water；
Second reagent, including 10~30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water；
3rd reagent, including 10 × Klenow polymerizations buffer solution, adenine, Klenow polymerases and ultra-pure water；
4th reagent, including 2 × DNA connections buffer solution, DNA ligase, Illumina sequence measuring joints and ultra-pure water；
5th reagent, including trishydroxymethylaminomethane, HCl and ultra-pure water；
6th reagent, including 2 × KAPA exo+ polymerases premixed liquid and Illumina sequencing primer mixed liquors.
Preferably, the kit also includes that PCR primer purifies magnetic bead.
Compared with prior art, advantages of the present invention includes：
(1) one kind that the present invention is provided is based on the coated trace dna sequencing pre-treating method of magnetic bead, is coated with using magnetic bead
DNA, effectively reduces the number of times that trace dna is transferred in pretreatment process is sequenced, so as to greatly reduce nucleic acid in sequencing
Loss in pretreatment process, does not use high temperature in whole processing procedure, reaction condition is gentle, while also reducing DNA damages
Wound, treatment trace dna sequencing pre-treatment success rate is improved to more than 90%, and more classical nucleic acid sequencing pre-treating method has
The characteristics of high success rate, high-quality, low initial amount；
(2) the trace dna sequencing pretreatment reagent kit of present invention offer, using the existing magnetic bead reagent in market and need to only divide
Sub- biological reagent, material source is simple, and operation difficulty is low, without know-how training and special instruments and equipment, compares market
Existing commercially available reagent box, simple and easy to get with low cost, library yield advantage high.
Brief description of the drawings
Fig. 1 is the production concentration contrast schematic diagram that embodiment of the present invention 1-4 and control group embodiment are obtained；
Fig. 2 is the cost consumption contrast schematic diagram of kit of the invention and kit A, kit B and classical way.
Specific embodiment
More detailed explanation will hereafter be made to technical scheme.It is understood, however, that in model of the present invention
In enclosing, can between above-mentioned each technical characteristic of the invention and each technical characteristic for specifically describing in below (eg embodiment)
It is combined with each other, so as to constitute new or preferred technical scheme.As space is limited, no longer tire out one by one herein and state.
Before being described to example, it is necessary to which some remarks explanations are provided：
The difference of experimental result can be caused using the reagent of different manufacturers, different batches, belongs to normal phenomenon.Carry out it is small
During sweeping experiment, to ensure the repeatability between parallel laboratory test, it is proposed that after configuration reagent, fully mix and dispense, to ensure every time
The homogeneity of experiment reagent.
The embodiment of the invention provides a kind of coated trace dna of magnetic bead that is based on and pre-treating method is sequenced, it includes：
Pending DNA is first carried out into magnetic bead coating, the coated pending DNA of magnetic bead end reparation is carried out into thereafter, then
Purifying, leaves magnetic bead, carries out plus A reactions, and repurity afterwards leaves magnetic bead, is attached reaction, afterwards repurity, abandons magnetic
Pearl, enters performing PCR reaction, after the completion of PCR reactions, adds magnetic bead and is purified, the DNA after being processed.
Among some embodiments, when carrying out magnetic bead coating, the pending DNA is with the volumetric usage ratio of magnetic bead
0.5~1.2:1.5~2.0.
Further, the magnetic bead is AmpureXP magnetic beads.
Among some embodiments, the liquor capacity of the pending DNA is 30uL~100uL；Preferably, the magnetic
The consumption of the adopted AmpureXP magnetic beads of pearl bag is 50uL~200uL；Preferably, after the completion of PCR reactions, the AmpureXP
The consumption of magnetic bead is 50uL~200uL.
Among some highly preferred embodiments, the consumption of the pending DNA is 10ng, and liquor capacity is
50uL；Preferably, the consumption of the adopted AmpureXP magnetic beads of the magnetic bead bag is 150uL；Preferably, after the completion of PCR reactions,
The consumption of the AmpureXP magnetic beads is 50uL.
Among some embodiments, the pre-treating method also includes：To adding in the coated pending DNA of magnetic bead
To carry out end reparation, first reagent is included one reagent：10X T4 DNA connection buffer solution, bovine serum albumin, ATP,
DNTPs, T4 phosphorylating kinase, T4 archaeal dna polymerases, Klenow Large fragment polymerases and ultra-pure water.
Further, the temperature program of the end reparation is：5min~20min is incubated at prior to 10 DEG C~15 DEG C, it
After 5min~20min is incubated at 20 DEG C~30 DEG C, most after 4 DEG C of preservations.
Further, the pH of first reagent is 7.3~8.5, and first reagent is included：10X T4 DNA connections are slow
The parts by volume of fliud flushing 2~20, the parts by volume of 1mg/mL bovine serum albumen solutions 2~10, the parts by volume of 10mM ATP solution 2~10,10mM
The parts by volume of dNTPs solution 1~5, the parts by volume of T4 phosphorylating kinases solution 2~10, the parts by volume of T4 archaeal dna polymerases solution 2~10,
The parts by volume of Klenow Large fragment polymerases solution 0.5~1.5, the parts by volume of ultra-pure water 10~50.
Further, the temperature program of the end reparation is：Be incubated 15min at prior to 12 DEG C, after at 25 DEG C
Insulation 15min, most after 4 DEG C of preservations.
It is more highly preferred to, the pH of first reagent is 8.0, and first reagent is included：10X T4 DNA connection bufferings
The parts by volume of liquid 5, the parts by volume of 1mg/mL bovine serum albumen solutions 5, the parts by volume of 10mM ATP solution 5, the volume of 10mM dNTPs solution 2
Part, the parts by volume of T4 phosphorylating kinases solution 5, the parts by volume of T4 archaeal dna polymerases solution 5, the body of Klenow Large fragment polymerases solution 1
Product part, the parts by volume of ultra-pure water 22.
Among some embodiments, the pre-treating method is additionally included in be carried out plus A reactions are preceding adds the 3rd reagent, institute
The 3rd reagent is stated to include：10 × Klenow polymerizations buffer solution, adenine, Klenow polymerases and ultra-pure water.
Specifically, the temperature program of described plus A reactions is：At prior to 30 DEG C~40 DEG C be incubated 5min~60min, after
4 DEG C of preservations；
Further, the pH of the 3rd reagent is 7.3~8.5, and the 3rd reagent is included：10 × Klenow polymerizations are slow
The parts by volume of fliud flushing 2~10, the parts by volume of 1mM adenine solutions 2~20, the parts by volume of exo-Klenow polymerase solutions 2~10 and super
The parts by volume of pure water 20~50.
More specifically, the temperature program for adding A to react is：Be incubated 30min at prior to 37 DEG C, after 4 DEG C preservation.
It is more highly preferred to, the pH of the 3rd reagent is 8.0, and the 3rd reagent is included：10 × Klenow polymerization bufferings
The parts by volume of liquid 5, the parts by volume of 1mM adenine solutions 10, the parts by volume of exo-Klenow polymerase solutions 3 and the parts by volume of ultra-pure water 32.
Among some embodiments, the pre-treating method is additionally included in before being attached reaction and adds the 4th reagent,
4th reagent is included：2 × DNA connections buffer solution, DNA ligase, Illumina sequence measuring joints and ultra-pure water.
Preferably, the temperature program of the coupled reaction is：5min~20min is incubated at prior to 20 DEG C~30 DEG C, afterwards
In 4 DEG C of preservations.
Further, the pH of the 4th reagent is 7.3~8.5, and the 4th reagent is included：2 × DNA connects buffer solution
5~25 parts by volume, the parts by volume of DNA ligase solution 1~5, the parts by volume of Illumina sequence measuring joints solution 0.5~3 and ultra-pure water
10~50 parts by volume.
More preferred, the temperature program of the coupled reaction is：Be incubated 15min at prior to 25 DEG C, after 4 DEG C guarantor
Deposit.
It is more highly preferred to, the pH of the 4th reagent is 8.0, and the 4th reagent is included：2 × DNA connects buffer solution
12.5 parts by volume, the parts by volume of DNA ligase solution 2.5, the parts by volume of Illumina sequence measuring joints solution 1 and the volume of ultra-pure water 34
Part.
Among some embodiments, the pre-treating method also includes：After abandoning magnetic bead, add before entering performing PCR reaction
Entering the 5th reagent carries out DNA wash-outs, is reacted to adding the 6th reagent to enter performing PCR in the DNA after wash-out.
Wherein, the 5th reagent is included：Trishydroxymethylaminomethane (Tris), HCl and ultra-pure water, the 6th reagent
Comprising：2 × KAPA HiFi exo+ polymerases premixed liquid (Ready Mix) and Illumina sequencing primer mixed liquors (Primer
Mix)。
It is more highly preferred to, the temperature program of the PCR reactions is：At a temperature of prior to 90 DEG C~100 DEG C preheat 1min~
5min, then carries out 2~20 circulations, and each circulation includes：Successively at 90 DEG C~100 DEG C be incubated 10s~50s, 55 DEG C~
At 72 DEG C be incubated 10s~50s, at 65 DEG C~85 DEG C be incubated 10s~50s, thereafter, at 60 DEG C~80 DEG C be incubated 1min~
10min, 4 DEG C of preservations.
Preferably, the pH of the 5th reagent is 7.3~8.5, and the consumption of the 5th reagent is 6uL~20uL, three hydroxyls
The concentration of aminomethane (Tris) is 5mM-20mM.
Further, the pH of the 6th reagent is 7.3~8.5, and the 6th reagent is included：2 × KAPA HiFi guarantors high
True polymerization enzyme premixed liquid (Ready Mix) 5~25 parts by volume, Illumina sequencing primers mixed liquor (Primer Mix) 1~5 body
Product part.
More specific, the temperature program of the PCR reactions is：2min is preheated at a temperature of prior to 95 DEG C, 7 are then carried out
Circulation, each circulation includes：30s is incubated at 95 DEG C successively, 30s is incubated at 65 DEG C, 30s is incubated at 72 DEG C, thereafter, at 72 DEG C
Lower insulation 4min, 4 DEG C of preservations.
Further, the pH of the 5th reagent is 8.0, and the consumption of the 5th reagent is 11uL, trihydroxy methyl amino
The concentration of methane (Tris) is 10mM.
Further, the pH of the 6th reagent is 8.0, and the 6th reagent is included：2 × KAPA HiFi high-fidelities are gathered
Synthase premixed liquid (Ready Mix) 12.5 parts by volume, Illumina sequencing primers mixed liquor (Primer Mix) 1.5 parts by volume.
It is of the invention based on the coated trace dna sequencing pre-treating method tool of magnetic bead among certain preferred embodiments
Body includes：
(1) the pending fragmentation DNA moisturizings of 10ng are taken to suitable volumes, and adds the AmpureXP magnetic beads to carry out magnetic bead bag
Quilt；
(2) to adding the first reagent to carry out end reparation in the magnetic bead being coated with；
(3) treat that end is repaired to complete, add the second reagent to be purified in single step reaction liquid forward；
(4) after the completion of purifying, magnetic bead is left, adds the 3rd reagent to carry out plus A reactions；
(5) after the completion of A reactions to be added, the second reagent is added to be purified in single step reaction liquid forward；
(6) after the completion of purifying, magnetic bead is left, adds the 4th reagent to be attached reaction；
(7) after the completion of reaction to be connected, purified to the second reagent is added in reaction solution；
(8) magnetic bead is abandoned after the completion of purifying, adds the 5th reagent to carry out DNA wash-outs, the DNA of wash-out is transferred to new PCR
Guan Zhong, adds the 6th reagent to enter performing PCR reaction；
(9) wait after the completion of PCR reactions, add AmpureXP magnetic beads to be purified, and eluted using the 5th reagent, obtain
End-product.
Wherein, the second reagent is added in the purification step, the pH of second reagent is 7.3~8.5, described second
Reagent is included：10~30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water.
It is more highly preferred to, the pH of second reagent is 8.0, and second reagent is included：20wt% PEG 8000s,
2.5M sodium chloride and ultra-pure water.
More specifically, the addition volume of the second reagent is 80uL~150uL in step (3), in the step (5), second
The composition of reagent is identical with step (3), adds volume also identical with step (3).In the step (7), the second reagent
Composition is identical with step (3), and it is 10uL~50uL, more preferably 35uL to add reagent volume consumption.
It is preferable that in the step (8), the pH of the 5th reagent is 8.0, and solvent is ultra-pure water, and solute is final concentration
Following material：, for adjusting pH, the amount of reagent is 11uL for trishydroxymethylaminomethane 10mM, HCl.
It is preferable that in the step (9), the composition of the 5th reagent is identical with step (8), addition volume is 10uL
~30uL, preferably 25uL.
The embodiment of the present invention additionally provides a kind of trace dna sequencing pretreatment reagent kit, and it is included：
First reagent, including 10X T4 DNA connection buffer solution, bovine serum albumin, ATP, dNTPs, T4 phosphorylating kinase,
T4 archaeal dna polymerases, Klenow Large fragment polymerases and ultra-pure water；
Second reagent, including 10~30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water；
3rd reagent, including 10 × Klenow polymerizations buffer solution, adenine, Klenow polymerases and ultra-pure water；
4th reagent, including 2 × DNA connections buffer solution, DNA ligase, Illumina sequence measuring joints and ultra-pure water；
5th reagent, including trishydroxymethylaminomethane (Tris), HCl and ultra-pure water；
6th reagent, including 2 × KAPA HiFi exo+ polymerases premixed liquid (Ready Mix) and Illumina sequencings
Primer mixed liquor (Primer Mix).
Preferably, the kit also includes that PCR primer purifies magnetic bead.
Below in conjunction with the technical solution of the present invention is further explained the explanation of accompanying drawing and some embodiments.
Embodiment 1
1. reagent prepares：
Ampure XP Beads (Beckman), absolute ethyl alcohol (traditional Chinese medicines), PEG 8000 (Sigma), sodium chloride (state
Medicine), Tris (traditional Chinese medicines), HCl (traditional Chinese medicines), Qubit dsDNA HS Assay (Life Technologies), 10X T4 DNA connect
Buffer solution (NEB) is connect, bovine serum albumin (1mg/mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylations swash
Enzyme (NEB), T4 archaeal dna polymerases (NEB), Klenow Large fragment polymerases (NEB), 10 × Klenow polymerizations buffer solution (NEB),
Adenine (1mM, NEB), Klenow polymerases (exo-, NEB), 2 × DNA connections buffer solution (NEB), DNA ligase (NEB),
Illumina sequence measuring joints (Bioo Scientific), 2 × KAPA HiFi Ready Mix (KAPA Biosystem),
Illumina Primer Mix(IDT)。
Reagent I：PH is 8.0, and constituent is：10X T4 DNA connect buffer solution 5uL, bovine serum albumin (1mg/mL)
5uL, ATP (10mM) 5uL, dNTPs (10mM) 2uL, T4 phosphorylating kinase 5uL, T4 archaeal dna polymerase 5uL, Klenow large fragment
Polymerase 1uL, ultra-pure water 22uL.
Reagent II：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：PEG 8000 20%
(w/v), sodium chloride 2.5M.
Reagent III：PH is 8.0, and constituent is：10 × Klenow polymerization buffer solution 5uL, adenine (1mM) 10uL,
Klenow polymerases (exo-) 3uL, ultra-pure water 32uL.
Reagent IV：PH is 8.0, and constituent is：2 × DNA connects buffer solution 12.5uL, DNA ligase 2.5uL,
Illumina sequence measuring joints 1uL, ultra-pure water 34uL.
Reagent V：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：Tris 10mM, HCl are for adjusting
Section pH.
Reagent VI：PH is 8.0, and constituent is：2 × KAPA HiFi Ready Mix 12.5uL, Illumina
Primer Mix 1.5uL。
2. experimental implementation process：
(1) take 10ng fragmentations DNA to be added in new 200ul PCR pipes, mend ultra-pure water to 50ul；
(2) to adding 150ul AMPure XP magnetic beads rifle to mix in 50ul fragmentations DNA more than 10 times, it is stored at room temperature
5min；
(3) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(4) washed 2 times with 80% ethanol of fresh configuration；
(5) drying at room temperature 3min, adds 50ul reagents I immediately；
(6) following procedure is run in PCR instrument：
| 12℃ | 15min |
| 25℃ | 15min |
| 4℃ | Forever |
(7) 110ul reagent IIs are added in repairing product to 50ul ends, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(8) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(9) washed 2 times with 80% ethanol of fresh configuration；
(10) drying at room temperature 3min, removes from magnetic frame, adds 50ul solvents III fully to mix magnetic bead；
(11) A response procedures are added below operation in PCR instrument：
| 37℃ | 30min |
| 4℃ | Forever |
(12) 110ul reagent IIs are added in adding A product to 50ul, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(13) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(14) washed 2 times with 80% ethanol of fresh configuration；
(15) drying at room temperature 3min, removes from magnetic frame, adds 50ul reagent IV, fully mixes magnetic bead；
(16) it is incubated 15min at 25 DEG C；
(17) to 35ul reagent IIs are added in 50ul coupled reaction products, mixed more than 10 times with rifle, be stored at room temperature 5min；
(18) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(19) washed 2 times with 80% ethanol of fresh configuration；
(20) drying at room temperature 3min, removes from magnetic frame, adds 13ul reagents V fully to mix magnetic bead；
(21) place supreme limpid clear on magnetic frame, draw 11ul supernatants and do PCR reactions；
(22) to reagent VI is added in 11ul products, after fully mixing, PCR programs are run；
(23) to addition 25ul H in 25ul PCR primers2It is 50ul that O is mended to volume, adds 50ul Ampure XP magnetic
Pearl, is stored at room temperature 5min；
(24) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(25) washed 2 times with 80% ethanol of fresh configuration；
(26) drying at room temperature 3min, removes from magnetic frame, adds 22ul reagents V fully to mix magnetic bead；
(27) place supreme limpid clear on magnetic frame, draw 20ul supernatants, into new 1.5ml EP pipes, obtain final product whole product
Thing, and it is quantified using Qubit dsDNA HS Assay.
3. experimental result：
Final product concentration is obtained for 12.6ng/uL.
Embodiment 2
1. reagent prepares：
Ampure XP Beads (Beckman), absolute ethyl alcohol (traditional Chinese medicines), PEG 8000 (Sigma), sodium chloride (state
Medicine), Tris (traditional Chinese medicines), HCl (traditional Chinese medicines), Qubit dsDNA HS Assay (Life Technologies), 10X T4DNA connect
Buffer solution (NEB) is connect, bovine serum albumin (1mg/mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylations swash
Enzyme (NEB), T4 archaeal dna polymerases (NEB), Klenow Large fragment polymerases (NEB), 10 × Klenow polymerizations buffer solution (NEB),
Adenine (1mM, NEB), Klenow polymerases (exo-, NEB), 2 × DNA connections buffer solution (NEB), DNA ligase (NEB),
Illumina sequence measuring joints (Bioo Scientific), 2 × KAPA HiFi Ready Mix (KAPA Biosystem),
Illumina Primer Mix(IDT)。
Reagent I：PH is 8.0, and constituent is：10X T4 DNA connect buffer solution 5uL, bovine serum albumin (1mg/mL)
5uL, ATP (10mM) 5uL, dNTPs (10mM) 2uL, T4 phosphorylating kinase 5uL, T4 archaeal dna polymerase 5uL, Klenow large fragment
Polymerase 1uL, ultra-pure water 22uL.
Reagent II：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：PEG 8000 20%
(w/v), sodium chloride 2.5M.
Reagent III：PH is 8.0, and constituent is：10 × Klenow polymerization buffer solution 5uL, adenine (1mM) 10uL,
Klenow polymerases (exo-) 3uL, ultra-pure water 32uL.
Reagent IV：PH is 8.0, and constituent is：2 × DNA connects buffer solution 12.5uL, DNA ligase 2.5uL,
Illumina sequence measuring joints 1uL, ultra-pure water 34uL.
Reagent V：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：Tris 10mM, HCl are for adjusting
Section pH.
Reagent VI：PH is 8.0, and constituent is：2 × KAPA HiFi Ready Mix 12.5uL, Illumina
Primer Mix 1.5uL。
2. experimental implementation process：
(1) take 10ng fragmentations DNA to be added in new 200ul PCR pipes, mend ultra-pure water to 50ul；
(2) to adding 150ul AMPure XP magnetic beads rifle to mix in 50ul fragmentations DNA more than 10 times, it is stored at room temperature
5min；
(3) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(4) washed 2 times with 80% ethanol of fresh configuration；
(5) drying at room temperature 3min, adds 50ul reagents I immediately；
(6) following procedure is run in PCR instrument：
| 12℃ | 15min |
| 25℃ | 15min |
| 4℃ | Forever |
(7) 110ul reagent IIs are added in repairing product to 50ul ends, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(8) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(9) washed 2 times with 80% ethanol of fresh configuration；
(10) drying at room temperature 3min, removes from magnetic frame, adds 50ul solvents III fully to mix magnetic bead；
(11) A response procedures are added below operation in PCR instrument：
| 37℃ | 30min |
| 4℃ | Forever |
(12) 110ul reagent IIs are added in adding A product to 50ul, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(13) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(14) washed 2 times with 80% ethanol of fresh configuration；
(15) drying at room temperature 3min, removes from magnetic frame, adds 50ul reagent IV, fully mixes magnetic bead；
(16) it is incubated 15min at 25 DEG C；
(17) to 35ul reagent IIs are added in 50ul coupled reaction products, mixed more than 10 times with rifle, be stored at room temperature 5min；
(18) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(19) washed 2 times with 80% ethanol of fresh configuration；
(20) drying at room temperature 3min, removes from magnetic frame, adds 13ul reagents V fully to mix magnetic bead；
(21) place supreme limpid clear on magnetic frame, draw 11ul supernatants and do PCR reactions；
(22) to reagent VI is added in 11ul products, after fully mixing, PCR programs are run；
(23) to addition 25ul H in 25ul PCR primers2It is 50ul that O is mended to volume, adds 50ul Ampure XP magnetic
Pearl, is stored at room temperature 5min；
(24) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(25) washed 2 times with 80% ethanol of fresh configuration；
(26) drying at room temperature 3min, removes from magnetic frame, adds 22ul reagents V fully to mix magnetic bead；
(27) place supreme limpid clear on magnetic frame, draw 20ul supernatants, into new 1.5ml EP pipes, obtain final product whole product
Thing, and it is quantified using Qubit dsDNA HS Assay.
3. experimental result：
Final product concentration is obtained for 13.4ng/uL.
Embodiment 3
1. reagent prepares：
Ampure XP Beads (Beckman), absolute ethyl alcohol (traditional Chinese medicines), PEG 8000 (Sigma), sodium chloride (state
Medicine), Tris (traditional Chinese medicines), HCl (traditional Chinese medicines), Qubit dsDNA HS Assay (Life Technologies), 10X T4DNA connect
Buffer solution (NEB) is connect, bovine serum albumin (1mg/mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylations swash
Enzyme (NEB), T4 archaeal dna polymerases (NEB), Klenow Large fragment polymerases (NEB), 10 × Klenow polymerizations buffer solution (NEB),
Adenine (1mM, NEB), Klenow polymerases (exo-, NEB), 2 × DNA connections buffer solution (NEB), DNA ligase (NEB),
Illumina sequence measuring joints (Bioo Scientific), 2 × KAPA HiFi Ready Mix (KAPA Biosystem),
Illumina Primer Mix(IDT)。
Reagent I：PH is 8.0, and constituent is：10X T4 DNA connect buffer solution 5uL, bovine serum albumin (1mg/mL)
5uL, ATP (10mM) 5uL, dNTPs (10mM) 2uL, T4 phosphorylating kinase 5uL, T4 archaeal dna polymerase 5uL, Klenow large fragment
Polymerase 1uL, ultra-pure water 22uL.
Reagent II：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：PEG 8000 20%
(w/v), sodium chloride 2.5M.
Reagent III：PH is 8.0, and constituent is：10 × Klenow polymerization buffer solution 5uL, adenine (1mM) 10uL,
Klenow polymerases (exo-) 3uL, ultra-pure water 32uL.
Reagent IV：PH is 8.0, and constituent is：2 × DNA connects buffer solution 12.5uL, DNA ligase 2.5uL,
Illumina sequence measuring joints 1uL, ultra-pure water 34uL.
Reagent V：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：Tris 10mM, HCl are for adjusting
Section pH.
Reagent VI：PH is 8.0, and constituent is：2 × KAPA HiFi Ready Mix 12.5uL, Illumina
Primer Mix 1.5uL。
2. experimental implementation process：
(1) take 10ng fragmentations DNA to be added in new 200ul PCR pipes, mend ultra-pure water to 50ul；
(2) to adding 150ul AMPure XP magnetic beads rifle to mix in 50ul fragmentations DNA more than 10 times, it is stored at room temperature
5min；
(3) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(4) washed 2 times with 80% ethanol of fresh configuration；
(5) drying at room temperature 3min, adds 50ul reagents I immediately；
(6) following procedure is run in PCR instrument：
| 12℃ | 15min |
| 25℃ | 15min |
| 4℃ | Forever |
(7) 110ul reagent IIs are added in repairing product to 50ul ends, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(8) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(9) washed 2 times with 80% ethanol of fresh configuration；
(10) drying at room temperature 3min, removes from magnetic frame, adds 50ul solvents III fully to mix magnetic bead；
(11) A response procedures are added below operation in PCR instrument：
| 37℃ | 30min |
| 4℃ | Forever |
(12) 110ul reagent IIs are added in adding A product to 50ul, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(13) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(14) washed 2 times with 80% ethanol of fresh configuration；
(15) drying at room temperature 3min, removes from magnetic frame, adds 50ul reagent IV, fully mixes magnetic bead；
(16) it is incubated 15min at 25 DEG C；
(17) to 35ul reagent IIs are added in 50ul coupled reaction products, mixed more than 10 times with rifle, be stored at room temperature 5min；
(18) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(19) washed 2 times with 80% ethanol of fresh configuration；
(20) drying at room temperature 3min, removes from magnetic frame, adds 13ul reagents V fully to mix magnetic bead；
(21) place supreme limpid clear on magnetic frame, draw 11ul supernatants and do PCR reactions；
(22) to reagent VI is added in 11ul products, after fully mixing, PCR programs are run；
(23) to addition 25ul H in 25ul PCR primers2It is 50ul that O is mended to volume, adds 50ul Ampure XP magnetic
Pearl, is stored at room temperature 5min；
(24) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(25) washed 2 times with 80% ethanol of fresh configuration；
(26) drying at room temperature 3min, removes from magnetic frame, adds 22ul reagents V fully to mix magnetic bead；
(27) place supreme limpid clear on magnetic frame, draw 20ul supernatants, into new 1.5ml EP pipes, obtain final product whole product
Thing, and it is quantified using Qubit dsDNA HS Assay.
3. experimental result：
Final product concentration is obtained for 11.6ng/uL.
Embodiment 4
1. reagent prepares：
Ampure XP Beads (Beckman), absolute ethyl alcohol (traditional Chinese medicines), PEG 8000 (Sigma), sodium chloride (state
Medicine), Tris (traditional Chinese medicines), HCl (traditional Chinese medicines), Qubit dsDNA HS Assay (Life Technologies), 10X T4DNA connect
Buffer solution (NEB) is connect, bovine serum albumin (1mg/mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylations swash
Enzyme (NEB), T4 archaeal dna polymerases (NEB), Klenow Large fragment polymerases (NEB), 10 × Klenow polymerizations buffer solution (NEB),
Adenine (1mM, NEB), Klenow polymerases (exo-, NEB), 2 × DNA connections buffer solution (NEB), DNA ligase (NEB),
Illumina sequence measuring joints (Bioo Scientific), 2 × KAPA HiFi Ready Mix (KAPA Biosystem),
Illumina Primer Mix(IDT)。
Reagent I：PH is 8.0, and constituent is：10X T4 DNA connect buffer solution 5uL, bovine serum albumin (1mg/mL)
5uL, ATP (10mM) 5uL, dNTPs (10mM) 2uL, T4 phosphorylating kinase 5uL, T4 archaeal dna polymerase 5uL, Klenow large fragment
Polymerase 1uL, ultra-pure water 22uL.
Reagent II：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：PEG 8000 20%
(w/v), sodium chloride 2.5M.
Reagent III：PH is 8.0, and constituent is：10 × Klenow polymerization buffer solution 5uL, adenine (1mM) 10uL,
Klenow polymerases (exo-) 3uL, ultra-pure water 32uL.
Reagent IV：PH is 8.0, and constituent is：2 × DNA connects buffer solution 12.5uL, DNA ligase 2.5uL,
Illumina sequence measuring joints 1uL, ultra-pure water 34uL.
Reagent V：PH is 8.0, and solvent is ultra-pure water, and solute is the following material of final concentration：Tris 10mM, HCl are for adjusting
Section pH.
Reagent VI：PH is 8.0, and constituent is：2 × KAPA HiFi Ready Mix 12.5uL, Illumina
Primer Mix 1.5uL。
2. experimental implementation process：
(1) take 10ng fragmentations DNA to be added in new 200ul PCR pipes, mend ultra-pure water to 50ul；
(2) to adding 150ul AMPure XP magnetic beads rifle to mix in 50ul fragmentations DNA more than 10 times, it is stored at room temperature
5min；
(3) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(4) washed 2 times with 80% ethanol of fresh configuration；
(5) drying at room temperature 3min, adds 50ul reagents I immediately；
(6) following procedure is run in PCR instrument：
| 12℃ | 15min |
| 25℃ | 15min |
| 4℃ | Forever |
(7) 110ul reagent IIs are added in repairing product to 50ul ends, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(8) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(9) washed 2 times with 80% ethanol of fresh configuration；
(10) drying at room temperature 3min, removes from magnetic frame, adds 50ul solvents III fully to mix magnetic bead；
(11) A response procedures are added below operation in PCR instrument：
| 37℃ | 30min |
| 4℃ | Forever |
(12) 110ul reagent IIs are added in adding A product to 50ul, is mixed more than 10 times with rifle, be stored at room temperature 5min；
(13) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(14) washed 2 times with 80% ethanol of fresh configuration；
(15) drying at room temperature 3min, removes from magnetic frame, adds 50ul reagent IV, fully mixes magnetic bead；
(16) it is incubated 15min at 25 DEG C；
(17) to 35ul reagent IIs are added in 50ul coupled reaction products, mixed more than 10 times with rifle, be stored at room temperature 5min；
(18) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(19) washed 2 times with 80% ethanol of fresh configuration；
(20) drying at room temperature 3min, removes from magnetic frame, adds 13ul reagents V fully to mix magnetic bead；
(21) place supreme limpid clear on magnetic frame, draw 11ul supernatants and do PCR reactions；
(22) to reagent VI is added in 11ul products, after fully mixing, PCR programs are run；
(23) to addition 25ul H in 25ul PCR primers2It is 50ul that O is mended to volume, adds 50ul Ampure XP magnetic
Pearl, is stored at room temperature 5min；
(24) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(25) washed 2 times with 80% ethanol of fresh configuration；
(26) drying at room temperature 3min, removes from magnetic frame, adds 22ul reagents V fully to mix magnetic bead；
(27) place supreme limpid clear on magnetic frame, draw 20ul supernatants, into new 1.5ml EP pipes, obtain final product whole product
Thing, and it is quantified using Qubit dsDNA HS Assay.
3. experimental result：
Final product concentration is obtained for 15.6ng/uL.
Control group embodiment
1. reagent prepares：
Absolute ethyl alcohol (traditional Chinese medicines), KAPA LTP Library Prep Kit (KAPA Biosystem).
2. experimental implementation process：
(1) take 10ng fragmentations DNA to be added in new 200ul PCR pipes, benefit Nuclease water to 50ul, and according to
Following systems add reagents in above-mentioned fragmentation DNA；
| Fragmentation DNA | 50ul |
| KAPA End Repair Buffer(10×) | 7ul |
| KAPA End Repair Enzyme Mix | 5ul |
| water | 8ul |
| Total | 70ul |
(2) 20 DEG C are placed, 30min is incubated；
(3) to 120ul AMPure XP magnetic beads are added in 70ul end-filling products, mixed more than 10 times with rifle, room temperature
Stand 5min；
(4) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(5) washed 2 times with 80% ethanol of fresh configuration；
(6) drying at room temperature 3min, removes from magnetic frame, adds following reagents：
| KAPA A-Tailing Buffer(10×) | 5ul |
| KAPA A-Tailing Enzyme | 3ul |
| water | 42 |
| Total | 50 |
(7) 30 DEG C are placed, 30min is incubated；
(8) 90ul SPRI Solution are added in adding A products to 50ul, 5min is stood after fully mixing；
(9) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(10) washed 2 times with 80% ethanol of fresh configuration；
(11) drying at room temperature 3min, removes from magnetic frame, adds following reagents：
| 5×KAPA Ligation Buffer | 10ul |
| KAPA T4 DNA Ligase | 5ul |
| Adapter(20nM) | 5ul |
| water | 30ul |
| Total | 50ul |
(12) 20 DEG C are placed, 15min is incubated；
(13) to 50ul SPRI Solution are added in 50ul connection products, 5min is stood after fully mixing；
(14) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(15) washed 2 times with 80% ethanol of fresh configuration；
(16) drying at room temperature 3min, removes from magnetic frame, is eluted with 20ul buffer solutions EB；
(17) it is carried out according to the following table the configuration of PCR reaction systems：
| Connection product DNA | 20ul |
| qPCR Primer(12.5uM) | 3ul |
| 2×KAPA HiFi HotStart ReadyMix | 25ul |
| water | 2ul |
| Total | 50ul |
(18) PCR temperature conditionss set as follows：
(19) to 50ul AMPure XP magnetic beads are added in 50ul PCR primers, 5min is stood after fully mixing；
(20) place supreme limpid clear on magnetic frame, supernatant is abandoned in suction；
(21) washed 2 times with 80% ethanol of fresh configuration；
(22) drying at room temperature 3min, is removed from magnetic frame, adds 22ul EB wash-outs, and 3min is stood after mixing；
(23) place supreme limpid clear on magnetic frame, in absorption 20ul to new 1.5ml EP pipes, you can finally produced
Thing, is quantified using Qubit dsDNA HS Assay to end-product.
3. experimental result：
Final product concentration is obtained for 8.7ng/uL.
Fig. 1 is the production concentration contrast schematic diagram that embodiment of the present invention 1-4 and control group embodiment are obtained, and Fig. 2 is this hair
Bright kit and the cost consumption contrast schematic diagram of kit A, kit B and classical way.Wherein, kit A is：KAPA
HTP Library Preparation Kit (KAPA Biosystem), kit B are：NextFlex DNA Library
Preparation Kit (NEB), classical way is：TruSeq DNA Library Prep Kit V2(Illumina).
From above example 1-4 and Fig. 1-2 it is obvious that of the invention based on the coated trace dna sequencing of magnetic bead
Pre-treating method, agents useful for same composition is simple, easy to operate, with low cost, is not required to special installation, can effectively reduce micro core
The number of times that acid is transferred in pretreatment process is sequenced, so as to greatly reduce loss of the nucleic acid in pretreatment process is sequenced,
High temperature is not used in whole processing procedure, reaction condition is gentle, while also reducing DNA damage, treatment trace dna is surveyed
Sequence pre-treatment success rate is improved to more than 90%；More classical nucleic acid sequencing pre-treating method, there is a high success rate, high-quality,
The characteristics of low initial amount, and by compareing the existing commercially available reagent box in market, have with low cost, library yield advantage high.
It should be appreciated that above-described embodiment is only explanation technology design of the invention and feature, this is familiar with its object is to allow
The personage of item technology will appreciate that present disclosure and implement according to this that it is not intended to limit the scope of the present invention.It is all
According to the equivalent change or modification that spirit of the invention is made, should all be included within the scope of the present invention.
Claims (13)
1. a kind of based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that including：
Pending DNA is first carried out into magnetic bead coating, the coated pending DNA of magnetic bead is carried out into end reparation thereafter, then purified,
Magnetic bead is left, is carried out plus A reactions, repurity afterwards leaves magnetic bead, is attached reaction, afterwards repurity, abandons magnetic bead, enters
Performing PCR reacts, and after the completion of PCR reactions, adds magnetic bead and is purified, the DNA after being processed.
2. according to claim 1 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that including：
When carrying out magnetic bead coating, the pending DNA is 0.5~1.2 with the volumetric usage ratio of magnetic bead:1.5~2.0, and/or, it is described
Magnetic bead is AmpureXP magnetic beads.
3. it is according to claim 1 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that also to wrap
Include：To the first reagent is added in the coated pending DNA of magnetic bead to carry out end reparation, first reagent is included：10X T4
DNA connections buffer solution, bovine serum albumin, ATP, dNTPs, T4 phosphorylating kinase, T4 archaeal dna polymerases, the polymerization of Klenow large fragments
Enzyme and ultra-pure water.
4. it is according to claim 3 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that described
End repair temperature program be：At prior to 10 DEG C~15 DEG C be incubated 5min~20min, after at 20 DEG C~30 DEG C be incubated
5min~20min, most after 4 DEG C of preservations；
And/or, the pH of first reagent is 7.3~8.5, and first reagent is included：10X T4 DNA connections buffer solution 2~
20 parts by volume, the parts by volume of 1mg/mL bovine serum albumen solutions 2~10, the parts by volume of 10mM ATP solution 2~10,10mM dNTPs are molten
The parts by volume of liquid 1~5, the parts by volume of T4 phosphorylating kinases solution 2~10, the parts by volume of T4 archaeal dna polymerases solution 2~10, Klenow is big
The parts by volume of fragment polymerase solution 0.5~1.5, the parts by volume of ultra-pure water 10~50.
5. it is according to claim 3 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that also to wrap
Include and add the 3rd reagent, the 3rd reagent to include before carrying out plus A reacts：10 × Klenow polymerization buffer solution, adenine,
Klenow polymerases and ultra-pure water.
6. it is according to claim 5 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that described
Plus the temperature program of A reactions is：At prior to 30 DEG C~40 DEG C be incubated 5min~60min, after 4 DEG C preservation；
And/or, the pH of the 3rd reagent is 7.3~8.5, and the 3rd reagent is included：10 × Klenow polymerizations buffer solution 2~
10 parts by volume, the parts by volume of 1mM adenine solutions 2~20, the parts by volume of exo-Klenow polymerase solutions 2~10 and ultra-pure water 20~
50 parts by volume.
7. it is according to claim 5 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that also to wrap
The 4th reagent of addition before reaction is attached is included, the 4th reagent is included：2 × DNA connection buffer solution, DNA ligase,
Illumina sequence measuring joints and ultra-pure water.
8. it is according to claim 7 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that described
The temperature program of coupled reaction is：At prior to 20 DEG C~30 DEG C be incubated 5min~20min, after 4 DEG C preservation；
And/or, the pH of the 4th reagent is 7.3~8.5, and the 4th reagent is included：2 × DNA connects the body of buffer solution 5~25
Product part, the parts by volume of DNA ligase solution 1~5, the parts by volume of Illumina sequence measuring joints solution 0.5~3 and the body of ultra-pure water 10~50
Product part.
9. it is according to claim 7 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that also to wrap
Include：After abandoning magnetic bead, the 5th reagent is added to carry out DNA wash-outs before entering performing PCR reaction, to addition the 6th in the DNA after wash-out
Reagent enters performing PCR reaction；
Wherein, the 5th reagent is included：Trishydroxymethylaminomethane, HCl and ultra-pure water, the 6th reagent are included：2×
KAPA exo+ polymerases premixed liquid and Illumina sequencing primer mixed liquors.
10. it is according to claim 9 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that institute
State PCR reaction temperature program be：1min~5min is preheated at a temperature of prior to 90 DEG C~100 DEG C, 2~20 is then carried out and is followed
Ring, each circulation includes：10s~50s is incubated at 90 DEG C~100 DEG C successively, 10s~50s, 65 DEG C are incubated at 55 DEG C~72 DEG C
10s~50s is incubated at~85 DEG C, thereafter, 1min~10min, 4 DEG C of preservations is incubated at 60 DEG C~80 DEG C；
And/or, the pH of the 5th reagent is 7.3~8.5, and the concentration of trishydroxymethylaminomethane is in the 5th reagent
5mM-20mM；
And/or, the pH of the 6th reagent is 7.3~8.5, and the 6th reagent is included：2 × KAPA HiFi high fidelity polymerases
The parts by volume of enzyme premixed liquid 5~25, the parts by volume of Illumina sequencing primers mixed liquor 1~5.
11. is according to claim 1 based on the coated trace dna sequencing pre-treating method of magnetic bead, it is characterised in that institute
State and add in purification step the second reagent, the pH of second reagent is 7.3~8.5, and second reagent is included：10~
30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water.
12. a kind of trace dna sequencing pretreatment reagent kits, it is characterised in that include：
First reagent, including 10X T4 DNA connections buffer solution, bovine serum albumin, ATP, dNTPs, T4 phosphorylating kinase, T4
Archaeal dna polymerase, Klenow Large fragment polymerases and ultra-pure water；
Second reagent, including 10~30wt% PEG 8000s, 1M~4M sodium chloride and ultra-pure water；
3rd reagent, including 10 × Klenow polymerizations buffer solution, adenine, Klenow polymerases and ultra-pure water；
4th reagent, including 2 × DNA connections buffer solution, DNA ligase, Illumina sequence measuring joints and ultra-pure water；
5th reagent, including trishydroxymethylaminomethane, HCl and ultra-pure water；
6th reagent, including 2 × KAPA exo+ polymerases premixed liquid and Illumina sequencing primer mixed liquors.
13. kits according to claim 12, it is characterised in that also include：PCR primer purifies magnetic bead.
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Application publication date: 20170524 |