CN107354148A - A kind of method for efficiently building storehouse for minim DNA - Google Patents
A kind of method for efficiently building storehouse for minim DNA Download PDFInfo
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- CN107354148A CN107354148A CN201710708670.2A CN201710708670A CN107354148A CN 107354148 A CN107354148 A CN 107354148A CN 201710708670 A CN201710708670 A CN 201710708670A CN 107354148 A CN107354148 A CN 107354148A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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Abstract
A kind of method for efficiently building storehouse for minim DNA disclosed by the invention, comprises the following steps：(1) it is used for DNA end-fillings using ultrapure T4 archaeal dna polymerases and cuts flat with, improves end remediation efficiency；(2) replace Taq archaeal dna polymerases to add A tails for DNA ends using Klenow (3 ' 5 ' exo), improve and add A efficiency；(3) the A/T connections being used for using ultrapure T4 DNA ligases between DNA and joint, joint efficiency is improved；(4) expanded using efficient high-fidelity enzyme.The invention has the advantages that：For minim DNA sample, this method builds storehouse improved efficiency, and sample builds storehouse transformation efficiency ＞ 10%.
Description
Technical field
The invention belongs to high throughput sequencing technologies field, more particularly to a kind of method for efficiently building storehouse for minim DNA.
Background technology
For minim DNA sample (＜ 50ng), conventional NEB, KAPAbiosystems and Illumina's builds storehouse reagent
Box builds that storehouse transformation efficiency is relatively low, and such as wherein KAPAbiosystems KAPA hyper build storehouse kit pair and 25ng170bp
The storehouse transformation efficiency of building of DNA fragmentation be 2.21%-8.84%, the storehouse transformation efficiency of building for 50ng170bp DNA fragmentation is
4.43%-8.84%.For minim DNA sample, it is to improve to imitate low frequency analysis of variance in sequencing detection that height, which builds storehouse transformation efficiency,
One of key parameter of rate, thus develop improve minim DNA sample build storehouse transformation efficiency experimental technique it is particularly significant and necessary.
Innovation and creation content
On the basis of the technical problems to be solved by the invention aim to provide a kind of traditional sequencing library structure advantage of reservation,
Efficiency is improved by reducing to be lost, to improve the transformation efficiency to minim DNA sample original DNA copy, and can be allowed to efficient
For the side for being used for minim DNA and efficiently building storehouse in the sequencing library structure of the minim DNA samples such as cfDNA (circulation dissociative DNA)
Method.
The technical problems to be solved by the invention can be achieved through the following technical solutions：
A kind of method for efficiently building storehouse for minim DNA, comprises the following steps：
(1) it is used for DNA end-fillings using ultrapure T4DNA polymerases and cuts flat with, improves end remediation efficiency.It is ultrapure
T4DNA polymerases use the P7080L of Enzymatics Inc. productions, and its 5 ' -3 ' polymerase activity possessed can be viscous with filling-in 5 '
Property end, its 3 ' -5 ' 5 prime excision enzyme activity can cut flat with 3 ' cohesive ends, possess higher purity, and preferably activity ensures end
Heterogeneous DNA fragmentation can form homogeneous flat end；
(2) replace Taq archaeal dna polymerases to add A tails for DNA ends using Klenow (3 ' -5 ' exo-), improve plus A is imitated
Rate；Klenow (3 ' -5 ' exo-) uses the P7010-HC-L of Enzymatics Inc. productions, and it lacks 3 ' -5 ' outside to be engineered
The normal temperature archaeal dna polymerase of enzyme cutting activity and 5 ' -3 ' nick translation nucleases, it can be held in flat terminal DNA fragments 3 ' and add dA
Tail；
(3) the A/T connections being used for using ultrapure T4DNA ligases between DNA and joint, joint efficiency is improved；It is ultrapure
T4DNA ligases use the L6030-HC-L of Enzymatics Inc. productions, can be with 5 ' phosphate groups of high-efficiency enzyme promoted connection and 3 ' hydroxyls
Base forms phosphodiester bond, efficiently connects the sample dna fragment of dT tails joint and dA tails, and repairs lacking in sample dna fragment
Carve；
(4) expanded using efficient high-fidelity enzyme；
Above-mentioned each step is all carried out in same PCR pipe, using AMpure magnetic beads by taking pearl method (with-
Beads) each step product is reclaimed in purifying, reduces sample and changes pipe loss.
Compared with prior art, the invention has the advantages that：
For minim DNA sample, this method builds storehouse improved efficiency, and sample builds storehouse transformation efficiency ＞ 10%.
Brief description of the drawings
Fig. 1 is the biological analyser analysis result schematic diagram of plasma DNA sample 2100 of the present invention.
Fig. 2 is the biological analyser analysis result of institute's structure library 2100 by sample of plasma DNA of the present invention.
Embodiment
Below with 1-100ng fragmentations DNA (DNA fragmentation, DNA fragmentation, the plasma frees of enzymic digestion interrupted such as ultrasound
DNA fragmentation etc.) it is sample, the present invention is described in detail with this for building library detection analysis.
The method that storehouse is efficiently built for minim DNA, comprises the following steps：
1.ER (End Repair) end-filling：35ng plasma DNA (accompanying drawing one, plasma DNA are drawn by concentration
It is secondary by 340bp fragments of advocating peace of 170bp fragments to be, is free on the native gene DNA of blood circulation system) in 200ul
In PCR pipe, with PCR level water polishings to 43ul, add 5ul 10xER buffer solutions (500mM NaCl, 100mM Tris-HCl,
100mM MgCl2,10mM DTT, 25 DEG C of 7.9@of pH), add 2ul T4 archaeal dna polymerases, whirlpool shake 3 seconds mix, rotate from
Solution is gathered in ttom of pipe in 3 seconds by the heart, and incubation reaction is carried out in PCR instrument, and reaction sets such as table one：
Table one：The flat end reactions of DNA are set
| Reaction temperature | Reaction time |
| 4℃ | 1min |
| 37℃ | 30min |
| 70℃ | 15min |
| 4℃ | Maintain |
2.AT (dA tailing) plus dA tails：0.5ul dATP (10mM) and 2ul are added in above-mentioned reaction product
Klenow (exo-), whirlpool concussion mix 3 seconds, rotating centrifugal 3 seconds, start incubation reaction in PCR instrument, reaction condition is shown in Table
Two：
Table two：DNA fragmentation adds dA end reactions to set
| Reaction temperature | Reaction time |
| 4℃ | 1min |
| 37℃ | 30min |
| 4℃ | Maintain |
Purified after 3.ER-AT：1.8x (94.5ul) AmPure magnetic beads are added in above-mentioned reaction, are purified by purification step
DNA fragmentation, and with 20ul eluted dnas fragment (reservation magnetic bead)；
4.Ligation sample dna fragments-joint connection：3ul 10x connection buffer solutions are added in above-mentioned eluted product
(500mM Tris-HCl, 100mM MgCl2,50mM DTT, 10mM ATP, 25 DEG C of 7.6@of pH), be proportionally added into joint amount and
The ultrapure ligases of 1.5ul, with PCR level water polishings to 30ul, whirlpool concussion mixes 3 seconds, rotating centrifugal 3 seconds, in PCR instrument
Incubation reaction, reaction condition are shown in Table three：
Table three：Sample dna fragment-joint coupled reaction is set
| Reaction temperature | Reaction time |
| 4℃ | 1min |
| 25℃ | 20min |
| 4℃ | Maintain |
5. purify one after connection：1.0x (24ul) PEG/NaCl is added in above-mentioned reaction, DNA pieces are purified by purification step
Section, and with 30ul eluted dnas fragment (reservation magnetic bead)；
6. purify two after connection：1.0x (24ul) PEG/NaCl is added in above-mentioned reaction, DNA pieces are purified by purification step
Section, and with 20ul eluted dnas fragment (reservation magnetic bead)；
7.PCR amplification of DNA fragments：5ul primers (Illumina P5/P7 universal primers) are added in above-mentioned eluted product
With 25ul 2xKAPA thermal starting exo+ polymerase premixed liquids, whirlpool concussion mixes 3 seconds, rotating centrifugal 3 seconds, it is anti-to enter performing PCR
Should, reaction condition is shown in Table four：
Table four：
It is pure after 8.PCR：1.0x (24ul) PEG/NaCl is added in above-mentioned reaction, by purification step purifying DNA fragment, and
With 30ul eluted dna fragments, library construction is completed；
9. take the above-mentioned libraries of 1ul to be used for TBS-380 fluorescence photometers detectable concentration and 2100 biological analysers analysis Library Quality,
Remaining be stored in -20 DEG C it is standby.Detect that library total amount is in TBS-380 fluorescence photometers using picogreen fluorescent dyes
480ng, show that Library Quality is good according to 2100 analyzing biochips results, the conversion of this banking process is shown according to this result
Efficiency is：12.1%.
Storehouse efficiency calculation formula is built in this technology method is：
Wherein n is PCR amplification cycles numbers, and specific in this example is calculated as：
Claims (1)
- A kind of 1. method for efficiently building storehouse for minim DNA, it is characterised in that comprise the following steps：(1) it is used for DNA end-fillings using ultrapure T4DNA polymerases and cuts flat with, improves end remediation efficiency；Ultrapure T4DNA gathers Synthase use Enzymatics Inc. production P7080L, its 5 ' -3 ' polymerase activity possessed can with the cohesive end of filling-in 5 ', Its 3 ' -5 ' 5 prime excision enzyme activity can cut flat with 3 ' cohesive ends, possess higher purity, and preferably activity ensures that end is heterogeneous DNA fragmentation can form homogeneous flat end；(2) replace Taq archaeal dna polymerases to add A tails for DNA ends using Klenow (3 ' -5 ' exo-), improve and add A efficiency； Klenow (3 ' -5 ' exo-) uses the P7010-HC-L of Enzymatics Inc. productions, and to lack 3 ' -5 ' circumscribed to be engineered for it The normal temperature archaeal dna polymerase of enzymatic activity and 5 ' -3 ' nick translation nucleases, it can be held in flat terminal DNA fragments 3 ' and add dA tails；(3) the A/T connections being used for using ultrapure T4DNA ligases between DNA and joint, joint efficiency is improved；Ultrapure T4DNA connects The L6030-HC-L that enzyme uses Enzymatics Inc. productions is met, can be formed with 5 ' phosphate groups of high-efficiency enzyme promoted connection and 3 ' hydroxyls Phosphodiester bond, efficiently connects the sample dna fragment of dT tails joint and dA tails, and repairs incising in sample dna fragment；(4) expanded using efficient high-fidelity enzyme；Above-mentioned each step is all carried out in same PCR pipe, pure by taking pearl method (with-beads) using AMpure magnetic beads Change and reclaim each step product, reduce sample and change pipe loss.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409408A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for accurate detection of whole genome methylation sites by utilizing trace genome DNA (deoxyribonucleic acid) |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
| CN103361743A (en) * | 2013-07-23 | 2013-10-23 | 安诺优达基因科技(北京)有限公司 | DNA library construction method for prenatal diagnosis |
| WO2016082057A1 (en) * | 2014-11-25 | 2016-06-02 | 深圳华大基因研究院 | Method for constructing free dna sequencing library |
| CN106702497A (en) * | 2015-11-17 | 2017-05-24 | 安诺优达基因科技(北京)有限公司 | Kit for assay circulating DNA (deoxyribonucleic acid) in maternal peripheral blood and library creation method |
-
2017
- 2017-08-17 CN CN201710708670.2A patent/CN107354148A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409408A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for accurate detection of whole genome methylation sites by utilizing trace genome DNA (deoxyribonucleic acid) |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
| CN103361743A (en) * | 2013-07-23 | 2013-10-23 | 安诺优达基因科技(北京)有限公司 | DNA library construction method for prenatal diagnosis |
| WO2016082057A1 (en) * | 2014-11-25 | 2016-06-02 | 深圳华大基因研究院 | Method for constructing free dna sequencing library |
| CN106702497A (en) * | 2015-11-17 | 2017-05-24 | 安诺优达基因科技(北京)有限公司 | Kit for assay circulating DNA (deoxyribonucleic acid) in maternal peripheral blood and library creation method |
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