CN106319033A - Method for detecting chromosome abnormality and recombinant site DNA sequence - Google Patents
Method for detecting chromosome abnormality and recombinant site DNA sequence Download PDFInfo
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Abstract
The invention provides a new method for detecting the chromosome abnormality and a recombinant site DNA sequence. The recombinant approximate region of chromosome is analyzed under low-cost conditions through a manure present technology, and a Cas9 in vitro multi-site single strand cutting technology is used to effectively overcome the disadvantage of short NGS read length in order to directly use NGS platform sequencing and greatly reduce the sequencing cost, so inversion information and translocation information of the chromosome are determined, and the DNA sequence of a target region is accurately determined. Compared with traditional chromosome abnormality detection ways, the method provided by the invention allows more DNA sequence information to be obtained.
Description
Technical field
The present invention relates to a kind of method detecting chromosomal abnormality, particularly relate to one can low cost, quickly survey
Determine chromosomal abnormality and chromosomal abnormality recombination region DNA sequence the method finding recombination site.
Background technology
Birth defect has severely impacted population quality, all brings huge loss to country, society and family.
Chromosome structure and quantity are to cause the main reason of birth defect extremely, and chromosomal structural abnormality includes dyeing
Body transposition and chromosome inversion etc..
Chromosome translocation refers to the change of chromosome segment position.When transposition occurs in item chromosome, claim
For displacement or intrachromosomal translocation；And when transposition occurs between two homologies or nonhomologous chromosome, be referred to as
Interchromosomal translocation.
Chromosome inversion is owing to there occurs twice fracture on same chromosome, its DNA segment warp produced
Cross 180 degree reverse, reconnect after cause chromosome inversion.If inversion generation is at one of chromosome
On arm, referred to as paracentric inversion；If inversion contains centric region, the most referred to as pericentric inversion (Anthony J.F.
Griffiths et al., An Introduction to Genetic Analysis, the 8th edition).
For many years, karyotyping and fluorescence in situ hybridization technique (FISH) are main Chromosomal abnormality analysis
Technology, has certain cost advantage in terms of Chromosome recombination approximate region identifying, but prenatal diagnosis chromosome
Abnormal sensitivity only have 71.9% (Yang Lingyun et al., China is eugenic and Journal of Heredity, 2009,17 (9):
1-4).In recent years, along with high-flux sequence (Next Generation Sequencing, NGS) of future generation
Developing rapidly of technology, NGS has been widely used in the analysis of chromosomal abnormality, but due to
Higher eukaryotic chromosome exists substantial amounts of repetitive sequence, and the sequence of NGS is read long shorter, is processing dye
During the complicated cases such as colour solid transposition and inversion, the limited use of NGS.BioNano and OpGen company
Technology can help to provide the frame diagram of full-length genome, helpful for identifying the position of chromosomal abnormality.But
Current scheme is still difficult to quickly and cheaply find out the DNA sequence of chromosomal abnormality position.
Summary of the invention
Cannot quickly and cheaply check order Chromosome recombination (transposition, inversion etc.) site for prior art
The problem of DNA sequence, a kind of method that the invention provides new detection recombination site DNA sequence.
The method of the detection chromosome disorder that first aspect of the present invention is provided, including:
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, contrasts with normal chromosomal DNA sequence, it may be judged whether deposit
At chromosome disorder.
Second aspect of the present invention is to provide a kind of method of detection chromosome disorder, including:
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, contrasts with normal chromosomal DNA sequence, it may be judged whether deposit
At chromosome disorder；
If it is determined there is variation, determine the precise region of recombination site, measure the DNA of the recombination site of precise region
Sequence.
Third aspect of the present invention is to provide a kind of method detecting Chromosome recombination site DNA sequence, including:
Primary Location chromosome translocation approximate region,
Canonical sequence according to this region of Article 1 chromosome designs and prepares the sgRNA sequence of sequence,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region of recombination site, and measures precise region
The DNA sequence of recombination site.
The method of the detection interchromosomal translocation recombination site DNA sequence that the 4th aspect of the present invention is provided,
Including:
Primary Location Chromosome recombination approximate region,
Canonical sequence according to Article 1 Chromosome recombination approximate region designs and prepares first group of sgRNA sequence of sequence
Row,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region in Article 1 Chromosome recombination site, and
Measure the DNA sequence of the recombination site of precise region；
Canonical sequence according to Article 2 Chromosome recombination approximate region designs and prepares second group of sgRNA sequence of sequence
Row,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region in Article 2 Chromosome recombination site, and
Measure the DNA sequence of the recombination site of precise region.
In foregoing of the present invention, the implication of described " approximate region " is bright to those skilled in the art
True, the generally bigger region containing Chromosome recombination site, such as 1-10M region, more preferably 1-8M
Region, more preferably 1-5M region.
In foregoing of the present invention, the implication of described " precise region " is bright to those skilled in the art
True, the generally smaller area containing Chromosome recombination site, such as≤20kb, it is more preferably≤15kb,
More preferably≤10kb, more preferably≤5kb, more preferably≤1kb.
Wherein, in foregoing of the present invention, the method for Primary Location Chromosome recombination approximate region, can be choosing
From the technology such as karyotyping, FISH, BioNano.
In described Primary Location Chromosome recombination approximate region step, it is also possible to include sentencing of chromosomal abnormality type
Disconnected.
Wherein, in a kind of preferred embodiment of the method for the invention, all right after chromosomal DNA cuts
Including utilizing DNA nick translation technology to mix the step of base analogue in indentation, there, and at double-stranded DNA
Feature purification of single stranded DNA of base analogue is utilized after untiing.
Wherein, in foregoing of the present invention, the implication of described " base analogue " is for those skilled in the art
For be clear and definite, refer to that chemical constitution is similar with base content, normal base can be substituted insert DNA
Compound in molecule, such as 8-a word used for translation guanine, Ismipur, 2-aminopurine, 5-bromouracil, 5-fluorine
DNTP, Azide that uracil, 5-bromine deoxidation urine nucleoside, maleic acid hydrazide, acrylamido are modified are modified
DNTP that dNTP, Biotin that dNTP, digoxin are modified modifies, any one in 5-hydroxymethyl cytosine
Plant or several, the dGTP that more preferably Biotin modifies.
Wherein, in a kind of preferred embodiment of the method for the invention, single stranded DNA is carried out at NGS platform
After directed sequencing, it is also possible to include the step utilizing existing Protocols in Molecular Biology accurately to measure target sequence
Suddenly.Wherein, described existing Protocols in Molecular Biology can be round pcr and other order-checkings known
Technology.
The present invention by the prior art of comparative maturity, can analyze the substantially district of Chromosome recombination under the conditions of low cost
Territory, and utilize Cas9 external multiple spot strand cutting technique, read to grow the shortcomings such as shorter efficiently against NGS,
It is thus possible to directly utilize the order-checking of NGS platform, significantly reduce order-checking cost.
The inventive method can not only determine the information such as chromosome inversion, transposition, additionally it is possible to purpose region
DNA sequence accurately measures.Compared to traditional chromosomal abnormality detection means, the method is obtained in that
More DNA sequence information.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of chromosome inversion regional DNA sequencing methods in the embodiment of the present invention 1；
Fig. 2 is the schematic flow sheet of chromosome translocation regional DNA sequencing methods in the embodiment of the present invention 2.
Detailed description of the invention
Below in conjunction with the accompanying drawings, the method for detection recombination site DNA sequence of the present invention is carried out detailed Jie
Continue and describe,.
Embodiment 1:
(1) determine that the situation of chromosomal abnormality (includes by the technology such as classical karyotyping, FISH, BioNano
On that chromosome, which kind of abnormal, Position Approximate), fall as it is shown in figure 1, chromosomal abnormality is chromosome
Position.
Navigate to the region (such as the region of 1-5M) substantially in chromosome inversion site.
(2) for the sgRNA sequence of this region design sequence, and external preparation sgRNA is carried out.
(3) extracting chromosomal DNA；According to CRISPR technology, utilize single endonuclease digestion activity (such as D10A or H840A)
Cas9, in vitro chromosome is cut.
(4) inactivation Cas9 enzyme, utilizes DNA nick translation technology to mix base analogue (such as Biotin in indentation, there
The dGTP modified).
(5) DNA is interrupted at random；Double-strand is untied and becomes single stranded DNA, and utilize the feature of base analogue
Purification of single stranded DNA.
(6) NGS platform is utilized to carry out single stranded DNA directed sequencing.
(7) sequence analysis, locking recombination site precise region (as < 10kb).
(8) if (7) do not find the sequence of accurate recombination site yet, then can utilize existing on the basis of (7)
The Protocols in Molecular Biology (PCR, order-checking etc.) having maturation accurately measures target sequence.
Embodiment 2:
(1) determine that the situation of chromosomal abnormality (includes by the technology such as classical karyotyping, FISH, BioNano
On that chromosome, which kind of is abnormal, Position Approximate), as in figure 2 it is shown, chromosomal abnormality is interchromosomal
Transposition.
Navigate to the region (such as the region of 1-5M) substantially in chromosome inversion site.
(2) for the sgRNA sequence of Article 1 chromosome c1 region design sequence, and external preparation is carried out
sgRNA。
(3) extracting chromosomal DNA；According to CRISPR technology, utilize single endonuclease digestion activity (such as D10A or H840A)
Cas9, in vitro chromosome is cut.
(4) inactivation Cas9 enzyme, utilizes DNA nick translation technology to mix base analogue (such as Biotin in indentation, there
The dGTP modified).
(5) DNA is interrupted at random；Double-strand is untied and becomes single stranded DNA, and utilize the feature of base analogue
Purification of single stranded DNA.
(6) NGS platform is utilized to carry out single stranded DNA directed sequencing.
(7) sequence analysis, locking recombination site precise region (as < 10kb)；
(8) then for the sgRNA sequence of Article 2 chromosome c2 region design sequence, and external preparation is carried out
SgRNA, repeats the step of above-mentioned (2)-(7).
(9) if (7) or (8) are not found the sequence of accurate recombination site yet, then can be in (7) or (8)
On the basis of, utilize the Protocols in Molecular Biology (PCR, order-checking etc.) of existing maturation accurately to measure target sequence.
A kind of adaptive immunity that CRISPR/Cas9 is antibacterial and archeobacteria is formed during long-term evolution is prevented
Imperial, can be used to resist the virus of invasion and foreign DNA (Horvath, P., and Barrangou, R. (2010).
CRISPR/Cas,the immune system of bacteria and archaea.Science 327,
167–170).CRISPR/Cas9 system is by being incorporated into the fragment of invasion phage and plasmid DNA
In CRISPR, and corresponding CRISPR RNAs (crRNAs) is utilized to instruct the degraded of homologous sequence,
Thus immunity is provided, the operation principle of this system is that crRNA (CRISPR-derived RNA) passes through
Base pairing and tracrRNA (trans-activating RNA) combine formation tracrRNA/crRNA and are combined
Thing, this complex guides nuclease Cas9 albumen in the sequence target site shearing double-strand matched with crRNA
DNA.And by engineer's both RNA, can transform and form the sgRNA with guiding function
(short guide RNA), it is sufficient to guide Cas9 the fixed point of DNA is cut (Jinek, M.,
Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,J.A.,and Charpentier,E.(2012).
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity.Science 337,816–821)。
Cas9 contains two the unique active structures of HNH in the middle part of aminoterminal RuvC and protein,
And double-stranded DNA ripe at crRNA plays a role in shearing.HNH avtive spot in Cas9 is sheared
The complementary dna chain of crRNA, RuvC avtive spot is sheared incomplementarity chain, is eventually introduced DNA double chain and breaks
Split (DSB).Suddenly change (D10A and the HNH domain such as RuvC1 to one of them domain
H840A) after, the Cas9 after sudden change just lose correspondence vigor (Jinek, M., Chylinski, K.,
Fonfara,I.,Hauer,M.,Doudna,J.A.,and Charpentier,E.(2012).A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity.Science 337,816–821).At present, along with the breakthrough of extensive work, Cas9's is external
Cutting system the most highly developed (such as, the external technology of preparing of sgRNA is very convenient and NEB company
Sell Cas9 enzyme through starting).
According to above-described embodiment it can be seen that present invention have the advantage that
1, ripe due to Technical comparing such as current chromosome karyotype analysis, FISH, if having only to identify dyeing body weight
The approximate region of group, cost is relatively low.The sequencing technologies of NGS also comparative maturity, puts down if NGS can be directly utilized
Platform, then detection process is not only quick, and cheaply.In the present invention, Cas9 external multiple spot strand is make use of to cut
Cut technology, nick translation technology etc. and carry out single stranded DNA labelling, then introduce NGS sequencing technologies, greatly
Reduce order-checking cost, and the long shortcoming such as shorter can be read efficiently against NGS.
2, combining NGS sequencing technologies, the method can not only determine the information such as chromosome inversion, transposition, moreover it is possible to
Enough DNA sequence to purpose region accurately measure.Compared to traditional chromosomal abnormality detection means,
The method is obtained in that more DNA sequence information.
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention is also
It is not restricted to particular embodiments described above.To those skilled in the art, any the present invention is carried out
Equivalent modifications and substitute the most all among scope of the invention.Therefore, without departing from the spirit of the present invention and model
Enclose lower made impartial conversion and amendment, all should contain within the scope of the invention.
Claims (10)
1. the method detecting recombination site DNA sequence, it is characterised in that including:
Primary Location chromosome translocation approximate region,
Canonical sequence according to this region of chromosome designs and prepares the sgRNA sequence of sequence,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region of recombination site, and measures precise region
The DNA sequence of recombination site.
Method the most according to claim 1, it is characterised in that described approximate region is containing Chromosome recombination
The 1-10M region in site.
Method the most according to claim 1, it is characterised in that described precise region is containing Chromosome recombination
Site≤region of 20kb.
Method the most according to claim 1, it is characterised in that described Primary Location Chromosome recombination substantially district
In the step of territory, also include the judgement of chromosomal abnormality type.
Method the most according to claim 1, it is characterised in that also include profit after chromosomal DNA cuts
Mix the step of base analogue in indentation, there by DNA nick translation technology, and after double-stranded DNA is untied
Utilize feature purification of single stranded DNA of base analogue.
Method the most according to claim 1, it is characterised in that described base analogue is that Biotin modifies
dGTP。
7. according to the method described in any one in claim 1-6, it is characterised in that described method is detection dye
The method of transposition recombination site DNA sequence between colour solid, including:
Primary Location Chromosome recombination approximate region,
Canonical sequence according to Article 1 Chromosome recombination approximate region designs and prepares first group of sgRNA sequence of sequence
Row,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region in Article 1 Chromosome recombination site, and
Measure the DNA sequence of the recombination site of precise region；
Canonical sequence according to Article 2 Chromosome recombination approximate region designs and prepares second group of sgRNA sequence of sequence
Row,
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, determines the precise region in Article 2 Chromosome recombination site, and
Measure the DNA sequence of the recombination site of precise region.
8. one kind is detected the method whether chromosome exists variation, it is characterised in that including:
According to CRISPR technology, utilize the Cas9 enzyme of single endonuclease digestion activity that chromosomal DNA is cut,
Double-stranded DNA is untied into single stranded DNA,
NGS platform carries out single stranded DNA directed sequencing, contrasts with normal chromosomal DNA sequence, it may be judged whether deposit
At chromosome disorder.
Method the most according to claim 8, it is characterised in that also comprise the steps:
If it is determined there is variation, determine the precise region of recombination site, measure the DNA of the recombination site of precise region
Sequence.
Method the most according to claim 8 or claim 9, it is characterised in that also wrap after chromosomal DNA cuts
Include and utilize DNA nick translation technology to mix the step of base analogue in indentation, there, and in double-stranded DNA solution
Feature purification of single stranded DNA of base analogue is utilized after opening.
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