CN107217310A - A kind of library constructing method and kit detected for chromosome abnormality - Google Patents
A kind of library constructing method and kit detected for chromosome abnormality Download PDFInfo
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- CN107217310A CN107217310A CN201710650277.2A CN201710650277A CN107217310A CN 107217310 A CN107217310 A CN 107217310A CN 201710650277 A CN201710650277 A CN 201710650277A CN 107217310 A CN107217310 A CN 107217310A
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- 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
Abstract
The present invention provides a kind of library constructing method detected for chromosome abnormality, by the optimization to reaction system and reaction condition, realizes the library construction to a small amount of sample such as amniotic fluid, bleeding of the umbilicus, shortens the stand-by period that patient detects in clinical chromosome abnormality.
Description
Technical field
The invention belongs to field of gene detection, and in particular to a kind of to be used for what chromosome abnormality was detected suitable for a small amount of sample
Sequencing library constructing method and kit.
Background technology
Chromosome is the inhereditary material in nucleus, and the mankind have 23 pairs of chromosomes, wherein 22 pairs are autosome, 1 pair is
Sex chromosome.Chromosome abnormality is including chromosomal structural abnormality and numerical abnormality, the disease caused by chromosome abnormality, clinically
Chromosomal disorder is shown as, the incidence of disease about 1% in more than 50%, neonate is accounted in dying young in spontaneous abortion, stillborn foetus, morning, belongs to normal
The inborn defect seen.
Numerical abnormalities of chromosomes refers to many 1 or several chromosomes, many 1 or several chromosome segments, common chromosome
Numerical abnormality is 13 3 bodies, 18 3 bodies and trisomy 21.In pre-natal diagnosis, the technology that predominantly detects of chromosome abnormality includes：Core
Type analysis, Comparative genomic hybridization (arrayCGH), fluorescence in situ hybridization technique, but be due to tire in amniotic fluid or Cord blood
Youngster's inhereditary material is less, it is necessary to be amplified to inhereditary material.Such as karyotyping, this method need to pass through cell culture
Enriches fetal cells, take time and effort very much, and patient waits the cycle of report long.
In recent years, it is rapid with high-flux sequence of future generation (Next Generation Sequencing, NGS) technology
Development, because it has a high resolution, the advantage such as flux is big has been widely used in the analysis of chromosome abnormality at present.It is logical
Chromosome abnormality can be detected by crossing traditional banking process, can be also found that new variation, but this method usually requires that higher DNA samples
This initial amount (more than 100ng), when collecting sample limited amount and (such as clinical acquisitions are again without reality when can not obtain enough DNA
Test the amniotic fluid amplified again, bleeding of the umbilicus equal samples), tend not to be met the text of follow-up sequencing demands by existing banking process
Storehouse yield.If can realize without cell culture, but directly carry out building storehouse using a small amount of sample such as amniotic fluid, bleeding of the umbilicus and detect dye
Colour solid is abnormal, can greatly save experimental period and testing cost, it will have very wide application prospect.
The content of the invention
In view of above-mentioned problems of the prior art, amniotic fluid etc. can be applied to it is an object of the invention to provide one kind
The construction method and kit in the sequencing library for being used for chromosome abnormality detection of a small amount of sample.
The present inventor has made intensive studies to solve the above problems, and as a result finds by optimizing reaction system and reaction bar
Part, realizes the sequencing library for building chromosome abnormality detection suitable for a small amount of sample of amniotic fluid, so as to complete the present invention.
That is, the present invention includes：
1. a kind of library constructing method detected for chromosome abnormality, this method includes：
Step A：Sample gDNA is extracted, gDNA is obtained；
Step B：The gDNA is subjected to digestion processing, purifying obtains digestion products；
Step C：The digestion products are subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step D：The flat terminal DNA fragments are subjected to 3' ends and add A, 3' ends plus A DNA fragmentation is obtained；
Step E：A DNA fragmentation is added to carry out adjunction head at the 3' ends, purifying obtains the DNA fragmentation of adjunction head；
Step F：The DNA fragmentation of adjunction head is entered into performing PCR amplification, purifying obtains amplified production；
Wherein, the enzyme that the digestion processing described in step B is used is dsDNA Fragmentase；
End described in step C is repaired to be included using reagent：1 μ L T4DNA polymerases, 1 μ L T4 polynueleotide kinases,
5 μ 10 × polynueleotide kinases of L buffer solutions and 1 μ L dNTP；
3' ends add A to include using reagent described in step D：0.5 μ L Klenow fragments (3'-5'exo-), 2.5 μ L dATP
And 2.5 μ 10 × buffer solutions of L；
The amplimer of the amplifications of PCR described in step F includes：
Ann primer sequences (SEQ ID NO:1)：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'
Index primer sequences (SEQ ID NO:2)：
5'-CAAGCAGAAGACGGCATACGAGATANNNNNNNGTGACTGGAGTTC-3'；
Wherein, N is randomized bases.
2. the library constructing method according to item 1, wherein, sample is amniotic fluid, bleeding of the umbilicus, whole blood, tissue in the step A
In any one.
3. the library constructing method according to item 1, wherein, gDNA amount is 8~15ng in the step B.
4. the library constructing method according to item 1, wherein, the condition of the digestion processing of the step B is 37 DEG C 50 points
Clock.
5. the library constructing method according to item 1, wherein, the condition that the end of the step C is repaired is 20 DEG C 30 points
Clock.
6. the library constructing method according to item 1, wherein, the 3' ends of the step D add A reaction condition to be 37 DEG C 30
Minute.
7. the library constructing method according to item 1, wherein, the PCR amplification conditions of the step F are 94 DEG C of pre-degenerations 2
Minute, (94 DEG C of denaturation are annealed 30 seconds, 72 DEG C for 15 seconds, 62 DEG C to be extended 30 seconds) 17 circulations, 72 DEG C of extensions are preserved for 10 minutes, 4 DEG C.
8. a kind of library construction Kit detected for chromosome abnormality, it is used for any one of practical matter 1~7
Library constructing method, it includes：
The reagent handled for the digestion；
The reagent repaired for the end；
Add A reagent for the 3' ends；
Reagent for adjunction head；
The reagent expanded for the PCR；
Reagent for the purifying.
Invention effect
The library constructing method and kit provided by the present invention, successfully to a small amount of amniotic fluid, the Cord blood of clinical acquisitions
Sample carries out library construction, realizes the direct detection to a small amount of amniotic fluid, Cord blood sample, had both avoided the experiment of cell culture
It is time-consuming, time and the testing cost of chromosome abnormality detection have greatly been saved, the standard of chromosome abnormality testing result is improved
True property.
The embodiment of invention
The scientific and technical terminology referred in this specification has the implication identical implication being generally understood that with those skilled in the art,
It is defined if any definition of the conflict in this specification.
First, the present invention provides a kind of library constructing method detected for chromosome abnormality, and it includes：
Step A：Sample gDNA is extracted, gDNA is obtained；
Step B：The gDNA is subjected to digestion processing, purifying obtains digestion products；
Step C：The digestion products are subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step D：The flat terminal DNA fragments are subjected to 3' ends and add A, 3' ends plus A DNA fragmentation is obtained；
Step E：A DNA fragmentation is added to carry out adjunction head at the 3' ends, purifying obtains the DNA fragmentation of adjunction head；
Step F：The DNA fragmentation of adjunction head is entered into performing PCR amplification, purifying obtains amplified production；
Wherein, the enzyme that the digestion processing described in step B is used is dsDNA Fragmentase；
End described in step C is repaired to be included using reagent：1 μ L T4DNA polymerases, 1 μ L T4 polynueleotide kinases,
5 μ 10 × polynueleotide kinases of L buffer solutions and 1 μ L dNTP；
3' ends add A to include using reagent described in step D：0.5 μ L Klenow fragments (3'-5'exo-), 2.5 μ L dATP
And 2.5 μ 10 × buffer solutions of L；
The amplimer of the amplifications of PCR described in step F includes：
Ann primer sequences (SEQ ID NO:1)：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'
Index primer sequences (SEQ ID NO:2)：
5'-CAAGCAGAAGACGGCATACGAGATANNNNNNNGTGACTGGAGTTC-3'；
Wherein, N is randomized bases, and NNNNNNN is sequence label.Herein, sequence label can be used as different sample libraries
Mark, sequence label that different samples are used is different, and sample data is carried out to sequencing data according to sequence label after sequencing
Distinguish.
Preferably, sample is any one in amniotic fluid, bleeding of the umbilicus, whole blood, tissue in the step A.
In the present invention, the low initial amount refers to amount of DNA in 8~15ng.
Preferably, the amniotic fluid sample of the step A is uncultivated sample, and gDNA amount is 10ng.
Preferably, the step B digestion processing condition for 37 DEG C 50 minutes.
Preferably, the end repairing condition of the step C be 20 DEG C 30 minutes.
Preferably, the 3' ends of the step D add A reaction condition for 37 DEG C 30 minutes.
Preferably, step F PCR amplification conditions be 94 DEG C 2 minutes, (94 DEG C 15 seconds, 62 DEG C 30 seconds, 72 DEG C 30 seconds) 17
Circulation, 72 DEG C 10 minutes, 4 DEG C preservation.
Preferably, in library constructing method of the invention, between step B and step C, between step C and step D, step E
Also include product purification steps between step F, after step F.Inventor's discovery, between step D and step E not
Purified, last library yield can be improved.For the purification step of the present invention, those skilled in the present invention can be using biography
It is used to method, reagent or the device of purifying in construction in a systematic way storehouse carry out.
On the other hand, a kind of library construction Kit for chromosome abnormality detection of present invention offer is (of the invention
Kit), it can be used for the library constructing method for implementing the present invention.The kit includes：
The reagent handled for the digestion, such as dsDNA Fragmentase and corresponding endonuclease reaction buffer solution；
The reagent repaired for the end, such as T4DNA polymerases, T4 polynueleotide kinases, dNTP, 10 × poly
Nucleotide kinase enzyme buffer liquid etc.；
Add A reagent for the 3' ends, for example, lack the Klenow fragments and corresponding reaction buffering of 5 prime excision enzyme activity
Liquid etc.；
For the reagent of adjunction head, such as joint, T4DNA ligases and corresponding coupled reaction buffer solution；
The reagent expanded for the PCR, such as KAPA HiFi HotStart ReadMix, Ann consensus primer,
Index primers etc.；
For the reagent of the purifying, such as nucleic acid purification reagent (annoroad companies), AMPure XP purifying magnetic bead
(agencourt companies) etc..
Embodiment
More specific description is carried out to the present invention by the following examples.It should be appreciated that embodiment described herein is
It is of the invention not for limiting for explaining the present invention.
Embodiment 1
1. genome (gDNA) is extracted
Two parts of amniotic fluid samples are taken, sample 1, sample 2 is respectively designated as, respectively take 2mL to use blood/tissue/cellular genome
Extracts kit (TIANGEN) carries out extracting genome DNA, obtains sample 1gDNA, sample 2gDNA.
2. digestion is handled
Sample 1 and sample 2 take respectively 10ng carry out digestion processing, single reaction system such as table 1 below,
Table 1
Reaction condition is 37 DEG C, 50 minutes；After reaction terminates, 5 μ L, 0.5M EDTA terminating reactions are added 2 minutes；Add
After 2.5 μ L 10%SDS are mixed, 49.5 μ L are taken to purify bead suspension (annoroad companies), 70% ethanol is washed twice, 43 μ L
ddH2O eluted dnas, obtain the digestion products of 1/ sample of sample 2.
3. end is repaired
The digestion products progress end reparation obtained to step 2, reaction system such as table 2 below,
Table 2
Reaction condition is 20 DEG C, 30 minutes, takes 75 μ L to purify bead suspension, 70% ethanol is washed twice, 20 μ L ddH2O
Eluted dna, obtains the flat terminal DNA fragments of 1/ sample of sample 2.
4.3' ends add A to react
The flat terminal DNA fragments obtained in step 3 are taken to carry out 3' ends plus A reactions by table 3, reaction system is as follows：
Table 3
Reaction condition is 37 DEG C, 30 minutes, obtains the DNA fragmentation that the 3' ends of 1/ sample of sample 2 add A.5. adjunction head reaction
Take the 3' ends obtained in step 4 plus A DNA fragmentation to carry out adjunction head to react, reaction system such as table 4 below,
Table 4
Reaction condition is 20 DEG C, 15 minutes, takes 78 μ L to purify bead suspension, 70% ethanol is washed twice, 18 μ LddH2O is washed
De- DNA, obtains the adjunction of 1/ sample of sample 2 head DNA fragmentation.
6.PCR reacts
The adjunction head DNA fragmentation obtained in step 5 is taken, enters performing PCR using the reaction system in table 5, reaction system is as follows
Table, sample 1 uses Index1 primers, and sample 2 uses Index2 primers,
Index1 primer sequences (SEQ ID NO:3)：
5'-CAAGCAGAAGACGGCATACGAGATAAGCAATGGTGACTGGAGTTC-3'
Index2 primer sequences (SEQ ID NO:4)：
5'-CAAGCAGAAGACGGCATACGAGATAATCCGAAGTGACTGGAGTTC-3'
Table 5
Reaction condition is：
45 μ L are taken to purify bead suspension, 70% ethanol is washed twice, 81 μ LddH2O eluted dnas, obtain the sample of sample 1/
2PCR amplified productions (library), complete library construction.
7. library concentration is determined
The peak figure and concentration in library are detected using Caliper and QPCR methods.
Analysis result such as table 6 below, the library yield of sample 1 is 470nmol/L, and the library yield of sample 2 is 695nmol/L, is said
Bright this method successfully builds storehouse, meets sequencing and requires.
Table 6
Embodiment 2
The library obtained in step 7 is sequenced using 550AR sequenators, sequencing strategy is SE50.By to data
It is analyzed, obtains sequencing data such as table 7 below, analysis sequencing data finds there is trisomy 21 in (such as table 8 below) sample 1,
There are 18 3 bodies in the amniotic fluid of sample 2.
In order to further prove the result of the present invention, sample 1 and sample 2 are analyzed using the method for karyotyping,
Analysis finds that sample 1 has trisomy 21, and sample 2 has 18 3 bodies, and the sequencing result one in library is as a result obtained with the inventive method
Cause.
Table 7
Table 8
It should also be noted that, on the premise of it can implement and substantially not run counter to the purport of the present invention, in this manual
It can also be equally applicable as the combination of any technical characteristic or technical characteristic described by the composition part of a certain technical scheme
In other technical schemes；Also, on the premise of it can implement and substantially not run counter to the purport of the present invention, it is used as different technologies scheme
Composition part described by technical characteristic between can also be combined in any way, to constitute other technical schemes.This
Invention be also contained in it is above-mentioned in the case of by technical scheme obtained from combination, and these technical schemes are equivalent to being documented in this
In specification.
The preferred embodiments of the present invention have shown and described in described above, as previously described, it should be understood that not office of the invention
Be limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations, modification and
Environment, and can be changed in invention contemplated scope described herein by the technology or knowledge of above-mentioned teaching or association area
It is dynamic., then all should be in the present invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of the present invention
In the protection domain of appended claims.
Industrial applicibility
In accordance with the invention it is possible to suitable for a small amount of sample such as amniotic fluid sample, chromosome abnormality detection library construction side
Method and kit.
Sequence table
<110>Pacify promise excellent up to Gene science（Beijing）Co., Ltd
<120>A kind of library constructing method and kit detected for chromosome abnormality
<130> 1701RGCN
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 45
<212> DNA
<213>Artificial sequence
<400>Ann primers
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC 45
<210> 2
<211> 45
<212> DNA
<213>Artificial sequence
<400>Index primers
CAAGCAGAAGACGGCATACGAGATANNNNNNNGTGACTGGAGTTC 45
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence
<400>Index1 primers
CAAGCAGAAGACGGCATACGAGATAAGCAATGGTGACTGGAGTTC 45
<210> 4
<211> 45
<212> DNA
<213>Artificial sequence
<400>Index2 primers
CAAGCAGAAGACGGCATACGAGATAATCCGAAGTGACTGGAGTTC 45
Claims (8)
1. a kind of library constructing method detected for chromosome abnormality, this method includes：
Step A：Sample gDNA is extracted, gDNA is obtained；
Step B：The gDNA is subjected to digestion processing, purifying obtains digestion products；
Step C：The digestion products are subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step D：The flat terminal DNA fragments are subjected to 3' ends and add A, 3' ends plus A DNA fragmentation is obtained；
Step E：A DNA fragmentation is added to carry out adjunction head at the 3' ends, purifying obtains the DNA fragmentation of adjunction head；
Step F：The DNA fragmentation of adjunction head is entered into performing PCR amplification, purifying obtains amplified production；
Wherein, the enzyme that the digestion processing described in step B is used is dsDNA Fragmentase；
End described in step C is repaired to be included using reagent：1 μ L T4 archaeal dna polymerases, 1 μ L T4 polynueleotide kinases, 5 μ L
10 × polynueleotide kinase buffer solution and 1 μ L dNTP；
3' ends add A to include using reagent described in step D：0.5 μ L Klenow fragments (3'-5'exo-), 2.5 μ L dATP and
2.5 μ 10 × buffer solutions of L；
The amplimer of the amplifications of PCR described in step F includes：
Ann primer sequences (SEQ ID NO:1)：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'
Index primer sequences (SEQ ID NO:2)：
5'-CAAGCAGAAGACGGCATACGAGATANNNNNNNGTGACTGGAGTTC-3'；
Wherein, N is randomized bases.
2. library constructing method according to claim 1, it is characterised in that in the step A sample be amniotic fluid, bleeding of the umbilicus,
Any one in whole blood, tissue.
3. library constructing method according to claim 1, it is characterised in that in the step B gDNA amount be 8~
15ng。
4. library constructing method according to claim 1, it is characterised in that the condition of the digestion processing of the step B is
37 DEG C 50 minutes.
5. library constructing method according to claim 1, it is characterised in that the condition that the end of the step C is repaired is
20 DEG C 30 minutes.
6. library constructing method according to claim 1, it is characterised in that the 3' ends of the step D add A reaction condition
For 37 DEG C 30 minutes.
7. library constructing method according to claim 1, it is characterised in that the PCR amplification conditions of the step F are 94 DEG C
Pre-degeneration 2 minutes, (94 DEG C be denatured to anneal for 15 seconds, 62 DEG C 30 seconds, 72 DEG C extend 30 seconds) 17 circulations, 72 DEG C extend 10 minutes, 4
DEG C preserve.
8. a kind of library construction Kit detected for chromosome abnormality, it is used to implement any one of claim 1~7 institute
The library constructing method stated, it includes：
The reagent handled for the digestion；
The reagent repaired for the end；
Add A reagent for the 3' ends；
Reagent for adjunction head；
The reagent expanded for the PCR；
Reagent for the purifying.
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