CN106282195A - Gene mutation body and application thereof - Google Patents
Gene mutation body and application thereof Download PDFInfo
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- CN106282195A CN106282195A CN201610665577.3A CN201610665577A CN106282195A CN 106282195 A CN106282195 A CN 106282195A CN 201610665577 A CN201610665577 A CN 201610665577A CN 106282195 A CN106282195 A CN 106282195A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
The invention discloses the nucleic acid of separation, the polypeptide of separation, screen the method for the biological sample that susceptible primary ciliary dyskinesia is levied, screen the system of the biological sample that susceptible primary ciliary dyskinesia is levied and for screening the test kit of the biological sample that susceptible primary ciliary dyskinesia is levied.Wherein, the nucleic acid of the coding DNA H5 of separation, there is compared with SEQ ID NO:1 c.G8030A sudden change, or the nucleic acid of the coding DNA H11 separated has c.C6265T sudden change compared with SEQ ID NO:3.By detecting whether these mutants exist in biological sample, can effectively detect the most susceptible primary ciliary dyskinesia of biological sample and levy.
Description
Technical field
The present invention relates to gene mutation body and application thereof.In particular it relates to separate nucleic acid, the polypeptide of separation,
Screen the method for the biological sample that susceptible primary ciliary dyskinesia is levied, screen the life that susceptible primary ciliary dyskinesia is levied
The system of thing sample, for screening the test kit of the biological sample that susceptible primary ciliary dyskinesia is levied, construct and weight
Group cell.
Background technology
Primary ciliary dyskinesia (primary ciliary dyskinesia, PCD), also known as the motionless syndrome of cilium
(immobile cilia syndrome), be because of ciliary structures defect cause moving obstacle cause cilium remove function reduce and
Other handicapped gene genetics are sick.This disease diverse clinical manifestations, can involve multiple organ-tissue, its diagnosis and differential diagnosis
Have difficulties.PCD present stage, find Disease-causing gene great to medical diagnosis on disease or therapeutic potential.Although having now been found that some PCD
Disease-causing gene, but focus rate is relatively low, and the PCD hypotype up to 18 reported at present, relates to tens of several gene, because of
This, there are still quite a few unknown Disease-causing gene site.
Thus, the research levied primary ciliary dyskinesia at present still needs deeply.
Summary of the invention
It is contemplated that at least solve one of technical problem present in prior art.To this end, one object of the present invention
Be to propose a kind of can the method for biological sample levied of the susceptible primary ciliary dyskinesia of Effective selection.
The present invention is following work based on inventor and completes: inventor is by the order-checking associating of high flux exon group
The method of candidate gene sudden change checking determines the new mutation on the Disease-causing gene that primary ciliary dyskinesia is levied.
According to the first aspect of the invention, the present invention proposes the nucleic acid of a kind of separation.According to embodiments of the invention, with
SEQ ID NO:1 compares, and this nucleic acid has c.G8030A sudden change；Or compared with SEQ ID NO:3, this nucleic acid has
C.C6265T suddenlys change.According to embodiments of the invention, inventor determines DNAH5 and DNAH11 gene mutation body, and these are newly dashed forward
The morbidity that variant and primary ciliary dyskinesia are levied is closely related, thus by detecting these new mutant in biological sample
Whether exist, can effectively detect the most susceptible primary ciliary dyskinesia of biological sample and levy.
According to the second aspect of the invention, the present invention proposes the polypeptide of a kind of separation.According to embodiments of the invention, with
SEQ ID NO:2 compares, and the polypeptide of this separation has p.R2677Q sudden change；Or compared with SEQ ID NO:4, this separation is many
Peptide has p.R2089* sudden change.By whether expressing this polypeptide in detection biological sample, can effectively detect biological sample is
No susceptible primary ciliary dyskinesia is levied.
According to the third aspect of the invention we, the present invention proposes and a kind of screens the life that susceptible primary ciliary dyskinesia is levied
The method of thing sample.According to embodiments of the invention, the method comprises the following steps: from extraction from biological material sample of nucleic acid；Really
The nucleotide sequence of fixed described sample of nucleic acid；The nucleotide sequence of described sample of nucleic acid or its complementary series, compared with SEQ ID NO:1
There is c.G8030A sudden change, or there is compared with SEQ ID NO:3 c.C6265T sudden change, be that described biological sample is susceptible primary
The instruction that the sex pili dyskinesia is levied.The life levied by the susceptible primary ciliary dyskinesia of screening according to embodiments of the present invention
The method of thing sample, can screen the biological sample that susceptible primary ciliary dyskinesia is levied effectively.
According to the fourth aspect of the invention, the present invention proposes and a kind of screens the life that susceptible primary ciliary dyskinesia is levied
The system of thing sample.According to embodiments of the invention, this system includes: nucleic acid-extracting apparatus, and described nucleic acid-extracting apparatus is used for
From described extraction from biological material sample of nucleic acid；Nucleotide sequence determines that device, described nucleotide sequence determine that device carries with described nucleic acid
Fetching is put connected, for being analyzed described sample of nucleic acid, in order to determine the nucleotide sequence of described sample of nucleic acid；Judge dress
Put, with described nucleotide sequence, described judgment means determines that device is connected, in order to nucleotide sequence based on described sample of nucleic acid or its
Complementary series, has c.G8030A sudden change compared with SEQ ID NO:1, or has c.C6265T compared with SEQ ID NO:3
Sudden change, it is judged that the most susceptible primary ciliary dyskinesia of described biological sample is levied.Utilize this system, it is possible to before effectively implementing
State the method screening the biological sample that susceptible primary ciliary dyskinesia is levied, such that it is able to it is fine effectively to screen susceptible constitutional
The biological sample that the hair dyskinesia is levied.
According to the fifth aspect of the invention, the present invention proposes and a kind of levies for screening susceptible primary ciliary dyskinesia
The test kit of biological sample.According to embodiments of the invention, this test kit contains: be adapted to detect for DNAH5 and DNAH11 gene
The reagent of at least one of mutant, wherein compared with SEQ ID NO:1, described DNAH5 gene mutation body has c.G8030A
Sudden change；Compared with SEQ ID NO:3, described DNAH11 gene mutation body has c.C6265T sudden change.Utilize according to the present invention's
The test kit of embodiment, it is possible to effectively screen the biological sample that susceptible primary ciliary dyskinesia is levied.
According to the sixth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this structure
Build body and comprise the nucleic acid of foregoing separation.It should be noted that the nucleic acid of foregoing separation " construct comprise " table
Showing, the construct of the present invention comprises the nucleic acid of the DNAH5 gene mutation body compared with SEQ ID NO:1 with c.G8030A sudden change
Sequence, or comprise the nucleotide sequence of the DNAH11 gene mutation body compared with SEQ ID NO:3 with c.C6265T sudden change, or
Person comprises above-mentioned DNAH5 gene mutation body and the nucleotide sequence of DNAH11 gene mutation body simultaneously.Thus, the structure of the present invention is utilized
Build the reconstitution cell that body transformed acceptor cell obtains, it is possible to be efficiently used for the medicine that screening treatment primary ciliary dyskinesia is levied
Thing.
According to the seventh aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, should
Reconstitution cell is obtained by foregoing construct transformed acceptor cell.According to some embodiments of the present invention, profit
With the reconstitution cell of the present invention, it is possible to the medicine that screening treatment primary ciliary dyskinesia is levied effectively.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage are from combining the accompanying drawings below description to embodiment and will become
Substantially with easy to understand, wherein:
Fig. 1: show and screen the biological sample that susceptible primary ciliary dyskinesia is levied according to an embodiment of the invention
The system of product and the schematic diagram of ingredient thereof, wherein,
A is showing of the system screening the biological sample that susceptible primary ciliary dyskinesia is levied according to the embodiment of the present invention
It is intended to,
B is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,
C is the schematic diagram that the nucleotide sequence according to the embodiment of the present invention determines device；
Fig. 2: show the family collection of illustrative plates of two PCD patient's familys according to an embodiment of the invention, wherein,
A is the family collection of illustrative plates of the PCD patient's family 1 according to the embodiment of the present invention,
B is the family collection of illustrative plates of the PCD patient's family 2 according to the embodiment of the present invention；
Fig. 3: show according to one embodiment of the invention, the proband in PCD patient's family 1 and the DNAH5 of father and mother thereof
The Sanger sequence verification peak figure of gene mutation site；
Fig. 4: show according to one embodiment of the invention, the proband in PCD patient's family 2 and father and mother's DNAH11 base thereof
Sanger sequence verification peak figure because of mutational site.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
Bright, and be not considered as limiting the invention.
DNAH5 and DNAH11 gene mutation body
According to the first aspect of the invention, the present invention proposes the nucleic acid of a kind of separation.According to embodiments of the invention, with
SEQ ID NO:1 compares, and this nucleic acid has c.G8030A sudden change；Or compared with SEQ ID NO:3, this nucleic acid has
C.C6265T suddenlys change.It should be noted that the nucleic acid coding DNAH5 of the separation of the present invention or DNAH11 mutant, it is also possible to
It is referred to as " coding DNA H5 or the nucleic acid of DNAH11 mutant ", i.e. this nucleic acid to can be understood as and coding DNA H5 or DNAH11
The nucleic acid substances that the gene of mutant is corresponding, i.e. the type of nucleic acid is not particularly limited, can be any comprise with DNAH5 and
Deoxyribonucleotide that the encoding gene of DNAH11 is corresponding and/or the polymer of ribonucleotide, include but not limited to
DNA, RNA or cDNA.A concrete example according to the present invention, foregoing coding DNA H5 and the core of DNAH11 mutant
Acid is DNA.According to embodiments of the invention, inventor determines the new mutant of DNAH5 and DNAH11 gene, this mutant with
The morbidity that primary ciliary dyskinesia is levied is closely related, thus by detecting whether this mutant exists in biological sample,
Can effectively detect the most susceptible primary ciliary dyskinesia of biological sample to levy, it is also possible to giving birth to by detecting this mutant
Whether object exists, can effectively predict that the most susceptible primary ciliary dyskinesia of organism is levied.
For, in description of the invention and claims, mentioning nucleic acid, it will be appreciated by those skilled in the art that actual bag
Include any one of complementary double-strand, or two.For convenience, in the present specification and claims, although most cases
Under only give a chain, but actually also disclose that another complementary therewith chain.Such as, mention SEQ ID NO:1, actual
Including its complementary series.Those skilled in the art are further appreciated that and utilize a chain can detect another chain, and vice versa.
These coding DNAs H5 and the nucleic acid of DNAH11 mutant, be that present inventor passes through high flux exon group
The method of order-checking associating candidate gene sudden change checking, newly dashing forward on the Disease-causing gene that the primary ciliary dyskinesia determined is levied
Become, and in the prior art and have not seen the c.G8030A sudden change on DNAH5 gene, and on DNAH11 gene
Relevant report is levied in c.C6265T sudden change to primary ciliary dyskinesia.
DNAH5 gene is antisense strand coding.The nucleotide sequence such as SEQ ID NO:1 of the cDNA of wild type DNAH5 gene
Shown (13875 nt):
Its coding aminoacid sequence (4624 aa) as shown in SEQ ID NO:2:
The nucleotide sequence of the cDNA of wild type DNAH11 gene is (13569 nt) as shown in SEQ ID NO:3:
Its coding aminoacid sequence (4523 aa) as shown in SEQ ID NO:4:
The DNAH5 gene mutation body that inventor finds, compared with SEQ ID NO:1, has c.G8030A sudden change, the most relatively
In wild type DNAH5 gene, the cDNA of the DNAH5 gene mutation body of the present invention, the G of the 8030th sports A, thus, and its institute
The product of coding, compared with wild type DNAH5 (SEQ ID NO:2), has p.R2677Q sudden change, and i.e. its 2677 amino acids is from R
Sport Q.
Additionally, the DNAH11 gene mutation body that inventor finds is compared with SEQ ID NO:3, there is c.C6265T sudden change,
I.e. relative to wild type DNAH11 gene, in the cDNA of the DNAH11 gene mutation body of the present invention, the C of the 6265th sports T,
Thus, its coded product, compared with wild type DNAH11, has p.R2089* sudden change, i.e. its 2089th coded amino acid
Termination codon, translation termination is become after the codon mutation of R.
The present inventor proposes c.G8030A sudden change and the c.C6265T of DNAH11 gene of DNAH5 gene first
Sudden change causes patient primary ciliary dyskinesia symptom occur.According to the second aspect of the invention, the present invention proposes one
The polypeptide separated.According to embodiments of the invention, compared with SEQ ID NO:2, the polypeptide of this separation has p.R2677Q sudden change；
Or compared with SEQ ID NO:4, the polypeptide of this separation has p.R2089* sudden change.According to some concrete examples of the present invention,
The polypeptide with p.R2677Q sudden change is by the nucleic acid coding of the coding DNA H5 mutant of aforementioned separation, has p.R2089*
The polypeptide of sudden change sudden change is by the nucleic acid coding of the coding DNA H11 mutant of aforementioned separation.By detection biological sample is
This polypeptide of no expression, can effectively detect the most susceptible primary ciliary dyskinesia of biological sample and levy, it is also possible to by inspection
Surveying whether these polypeptide exist in organism, the prediction the most susceptible primary ciliary dyskinesia of organism is levied effectively.
The method screening the biological sample that susceptible primary ciliary dyskinesia is levied
According to the third aspect of the invention we, the present invention proposes and a kind of screens the life that susceptible primary ciliary dyskinesia is levied
The method of thing sample.According to embodiments of the invention, the side of the biological sample that the susceptible primary ciliary dyskinesia of this screening is levied
Method may comprise steps of:
First, from extraction from biological material sample of nucleic acid.According to embodiments of the invention, the type of biological sample is not by spy
Do not limit, as long as can extract from this biological sample whether reflection biological sample DNAH5 and DNAH11 exists the core of sudden change
Acid sample.According to embodiments of the invention, biological sample can be selected from blood of human body, skin, hypodermic at least
A kind of.Thus, it is possible to be sampled easily and detect such that it is able to improve further and screen susceptible Primary Ciliary motion barrier
Hinder the efficiency of the biological sample levied.According to embodiments of the invention, term used herein above " sample of nucleic acid " should do broad sense reason
Solving, it can be any can to reflect in biological sample, whether DNAH5 and DNAH11 exists the sample of sudden change, can be such as from
The complete genome DNA of extracting directly in biological sample, it is also possible to be to comprise DNAH5 and DNAH11 coded sequence in this full-length genome
A part, can be from biological sample extract total serum IgE, it is also possible to be from biological sample extract mRNA.According to this
One embodiment of invention, described sample of nucleic acid is complete genome DNA.Thus, it is possible to expand the source range that comes of biological sample, and
And can the much information of biological sample be determined such that it is able to improve and screen susceptible primary ciliary dyskinesia. simultaneously
The efficiency of the biological sample levied.It addition, according to embodiments of the invention, for employing RNA as sample of nucleic acid, from biological sample
Extraction sample of nucleic acid may further include: from extraction from biological material RNA sample, preferably RNA sample is mRNA；And based on institute
The RNA sample obtained, passes through reverse transcription reaction, it is thus achieved that cDNA sample, and obtained cDNA sample constitutes sample of nucleic acid.Thus,
Can improve further and utilize RNA to screen the effect of the biological sample that susceptible primary ciliary dyskinesia is levied as sample of nucleic acid
Rate.
It follows that after obtaining sample of nucleic acid, sample of nucleic acid can be analyzed such that it is able to determine obtained core
The nucleotide sequence of acid sample.According to embodiments of the invention, the method and apparatus determining the nucleotide sequence of obtained sample of nucleic acid
It is not particularly restricted.According to a particular embodiment of the invention, sequence measurement can be passed through, determine the nucleic acid sequence of sample of nucleic acid
Row.According to embodiments of the invention, the method and apparatus that may be used for carrying out checking order is not particularly restricted.According to the present invention's
Embodiment, can use second filial generation sequencing technologies, it would however also be possible to employ the sequencing technologies of the third generation and forth generation or more advanced.
Concrete example according to the present invention, it is possible to use selected from Hiseq2000, SOLiD, 454 and at least the one of single-molecule sequencing device
Plant and nucleotide sequence is checked order.Thus, in conjunction with up-to-date sequencing technologies, higher order-checking can be reached for Single locus deep
Degree, detection sensitivity and accuracy are greatly improved, it is thus possible to utilize the spy that the high flux of these sequencing devices, the degree of depth check order
Point, improves further and sample of nucleic acid carries out the efficiency that detection is analyzed.Follow-up sequencing data is analyzed thus, it is possible to improve
Time accuracy and accuracy.Thus, according to embodiments of the invention, determine that the nucleotide sequence of sample of nucleic acid can wrap further
Include: first, for obtained sample of nucleic acid, build nucleic acid sequencing library；And obtained nucleic acid sequencing library is carried out
Order-checking, in order to obtain the sequencing result being made up of multiple sequencing datas.According to some embodiments of the present invention, can use and be selected from
Checked order in obtained nucleic acid sequencing library by least one of Hiseq2000, SOLiD, 454 and single-molecule sequencing device.
It addition, according to embodiments of the invention, sample of nucleic acid can be screened, enrichment DNA H5 and DNAH11 exon, this screening
Enrichment can be before building sequencing library, during building sequencing library, or carries out after structure sequencing library.According to this
One embodiment of invention, for sample of nucleic acid, builds nucleic acid sequencing library and farther includes: utilize selected from DNAH5 and
At least one of DNAH11 gene extron specific primer, carries out PCR amplification to sample of nucleic acid；And for obtained expansion
Volume increase thing, builds nucleic acid sequencing library.Thus, it is possible to expanded by PCR, enrichment DNA H5 and DNAH11 exon such that it is able to
Improve the efficiency screening the biological sample that susceptible primary ciliary dyskinesia is levied further.According to embodiments of the invention,
The sequence of DNAH5 and DNAH11 gene extron specific primer is not particularly limited, according to a preferred embodiment of the invention,
DNAH5 gene extron specific primer can have the nucleotide sequence as shown in SEQ ID NO:5-6, outside DNAH11 gene
Aobvious sub-specific primer can have the nucleotide sequence as shown in SEQ ID NO:7-8.It is surprisingly found by the inventors that, by adopting
With the primer shown in SEQ ID NO:5-6, significantly can effectively complete to DNAH5 especially in PCR reaction system
The amplification of the exon sequence at c.G8030A place；Use the primer shown in SEQ ID NO:7-8, can significantly effectively complete
Amplification to DNAH11 especially c.C6265T place exon sequence.It should be noted that shown in these SEQ ID NO:5-8
Nucleotide sequence be the present inventor after having paid arduous labor, unexpected obtain.
About for sample of nucleic acid, building method and the flow process of sequencing library, those skilled in the art can be according to difference
Sequencing technologies suitably select, about the details of flow process, the such as Illumina company of manufacturer of the instrument that may refer to check order
The code provided, for example, see Illumina company Multiplexing Sample Preparation Guide (Part#
1005361；Feb 2010) or Paired-End SamplePrep Guide (Part#1005063；Feb 2010), by ginseng
According to being incorporated into herein.According to embodiments of the invention, from the method and apparatus of extraction from biological material sample of nucleic acid, the most not by spy
Do not limit, the nucleic acid extraction kit of commercialization can be used to carry out.
It should be noted that the term here used " nucleotide sequence " should broadly understood, it can be to core
Acid sample carries out checking order after the sequencing data obtained assembles, the complete nucleic acid sequence information obtained, it is also possible to be direct
Use by the sequencing data (reads) that sample of nucleic acid is checked order obtained as nucleotide sequence, if these nucleic acid sequences
Containing corresponding DNA H5 and the coded sequence of DNAH11 gene in row.
Finally, after the nucleotide sequence determining sample of nucleic acid, by corresponding for the nucleotide sequence of obtained sample of nucleic acid
Reference sequences is compared, when obtained nucleotide sequence has aforesaid three kinds sudden change at least one time, i.e. indicate life
The susceptible primary ciliary dyskinesia of thing sample is levied.Specifically, it is to use DNAH5 gene extron special when the sample of nucleic acid obtained
When specific primer amplification obtains, by the sequence phase comparison of the nucleotide sequence of this sample of nucleic acid with SEQ ID NO:1, if at gained
To nucleotide sequence in have c.G8030A sudden change, it indicates that the susceptible primary ciliary dyskinesia of biological sample is levied.Work as acquisition
Sample of nucleic acid be use DNAH11 gene extron primer amplified obtain time, by the nucleotide sequence of this sample of nucleic acid with
The sequence phase comparison of SEQ ID NO:3, if having c.C6265T sudden change, it indicates that biological sample in obtained nucleotide sequence
The susceptible primary ciliary dyskinesia of product is levied.When the sample of nucleic acid obtained is to use DNAH11 and DNAH5 gene extron special
Property primer amplification obtain time, by the nucleotide sequence of this sample of nucleic acid respectively with SEQ ID NO:1 and the sequence phase of SEQ ID NO:3
Comparison, if having c.G8030A, c.C6265T sudden change, it indicates that biological sample is susceptible primary in obtained nucleotide sequence
The sex pili dyskinesia is levied.Thus, the life levied by the susceptible primary ciliary dyskinesia of screening according to embodiments of the present invention
The method of thing sample, can screen the biological sample that susceptible primary ciliary dyskinesia is levied effectively.Reality according to the present invention
Executing example, the method and apparatus comparing nucleotide sequence and SEQ ID NO:1 and SEQ ID NO:3 is not particularly restricted,
The software that can use any conventional operates, and according to the instantiation of the present invention, can use SOAPALIGNER/SOAP2
Compare.
It should be noted that according to embodiments of the present invention " screen the biological sample that susceptible primary ciliary dyskinesia is levied
The method of product " purposes be not particularly limited, such as can serve as the screening technique of non-diagnostic purpose.
Screen system and the test kit of the biological sample that susceptible primary ciliary dyskinesia is levied
According to the fourth aspect of the invention, the present invention proposes one can effectively to implement the susceptible constitutional of above-mentioned screening fine
The system of the method for the biological sample that the hair dyskinesia is levied.
With reference to Fig. 1, according to embodiments of the invention, the biological sample that the susceptible primary ciliary dyskinesia of this screening is levied
System 1000 includes: nucleic acid-extracting apparatus 100, nucleotide sequence determine device 200 and judgment means 300.
According to embodiments of the invention, nucleic acid-extracting apparatus 100 is for from extraction from biological material sample of nucleic acid.Such as front institute
Stating, according to embodiments of the invention, the type of sample of nucleic acid is not particularly restricted, for employing RNA as sample of nucleic acid, then
Nucleic acid-extracting apparatus farther includes RNA extraction unit 101 and reverse transcription unit 102, and wherein, extraction unit 101 is for from life
Thing sample extraction RNA sample, reverse transcription unit 102 is connected with RNA extraction unit 101, anti-for RNA sample is carried out reverse transcription
Should, in order to obtain cDNA sample, obtained cDNA sample constitutes sample of nucleic acid.
According to embodiments of the invention, nucleotide sequence determines that device 200 is connected with nucleic acid-extracting apparatus 100, for core
Acid sample is analyzed, in order to determine the nucleotide sequence of sample of nucleic acid.As previously shown, the method for order-checking can be used to determine nucleic acid
The nucleotide sequence of sample.Thus, according to one embodiment of present invention, described nucleotide sequence determines that device 200 can be further
Including: library construction unit 201 and order-checking unit 202.Library construction unit 201, for for sample of nucleic acid, builds nucleic acid
Sequencing library；Order-checking unit 202 is connected with library construction unit 201, for checking order nucleic acid sequencing library, in order to obtain
The sequencing result being made up of multiple sequencing datas.As it was previously stated, can be expanded by PCR, aobvious outside enrichment DNA H5 and DNAH11
Son, improves the efficiency screening the biological sample that susceptible primary ciliary dyskinesia is levied further.Thus, library construction unit
201 may further include PCR expands module (not shown), be provided with in this PCR amplification module selected from DNAH5 and
At least one of DNAH11 gene extron specific primer, in order to utilize DNAH5 and DNAH11 gene extron specificity to draw
At least one of thing, carries out PCR amplification, according to a particular embodiment of the invention, DNAH5 gene extron to described sample of nucleic acid
Specific primer has the nucleotide sequence as shown in SEQ ID NO:5-6, and DNAH11 gene extron specific primer has
Nucleotide sequence as shown in SEQ ID NO:7-8.According to embodiments of the invention, order-checking unit 202 can include being selected from
At least one of HISEQ2000, SOLiD, 454 and single-molecule sequencing device.Thus, in conjunction with up-to-date sequencing technologies, for list
Individual site can reach the higher order-checking degree of depth, and detection sensitivity and accuracy are greatly improved, it is thus possible to utilize these to check order
The feature of the high flux of device, degree of depth order-checking, improves further and sample of nucleic acid carries out the efficiency that detection is analyzed.Thus, improve
Follow-up accuracy time sequencing data is analyzed and accuracy.
According to embodiments of the invention, it is judged that device 300 determines that with nucleotide sequence device 200 is connected, and is suitable to nucleic acid sample
This nucleotide sequence is compared, in order to nucleotide sequence based on sample of nucleic acid and SEQ ID NO:1 and the district of SEQ ID NO:3
Do not judge that the most susceptible primary ciliary dyskinesia of biological sample is levied.Specifically, nucleotide sequence based on described sample of nucleic acid
Or its complementary series, there is compared with SEQ ID NO:1 c.G8030A sudden change, or have compared with SEQ ID NO:3
C.C6265T suddenlys change, it is judged that the most susceptible primary ciliary dyskinesia of biological sample is levied.As it was previously stated, according to the one of the present invention
Individual embodiment, the nucleotide sequence of sample of nucleic acid or its complementary series, have compared with SEQ ID NO:1 and dash forward selected from c.G8030A
Become, or there is compared with SEQ ID NO:3 c.C6265T sudden change, be that the susceptible primary ciliary dyskinesia of biological sample is levied
Instruction.As it was previously stated, according to embodiments of the invention, nucleotide sequence is compared with SEQ ID NO:1 and SEQ ID NO:3
To equipment be not particularly restricted, the software of any conventional can be used to operate, such as concrete real according to the present invention
Example, can use SOAPALIGNER/SOAP2 to compare.
Thus, this system is utilized, it is possible to effectively implement the biology that the susceptible primary ciliary dyskinesia of aforementioned screening is levied
The method of sample, such that it is able to effectively screen the biological sample that susceptible primary ciliary dyskinesia is levied.
According to the fifth aspect of the invention, the present invention proposes and a kind of levies for screening susceptible primary ciliary dyskinesia
The test kit of biological sample.According to embodiments of the invention, this is used for screening the life that susceptible primary ciliary dyskinesia is levied
The test kit of thing sample includes: be adapted to detect for the reagent of at least one of DNAH5 and DNAH11 gene mutation body, wherein with SEQ
ID NO:1 compares, and this DNAH5 gene mutation body has c.G8030A sudden change；Compared with SEQ ID NO:3, this DNAH11 gene
Mutant has c.C6265T sudden change.Utilize test kit according to an embodiment of the invention, it is possible to effectively screen susceptible primary
The biological sample that the sex pili dyskinesia is levied.In this article, the term used " is adapted to detect for DNAH5 and DNAH11 gene to dash forward
The reagent of at least one of variant " should be interpreted broadly, can be i.e. at least one of detection DNAH5 and DNAH11 encoding gene
Reagent, it is also possible to be detection DNAH5 and DNAH11 mutant the reagent of at least one, such as can use identification specificity
The antibody in site.According to one embodiment of present invention, this reagent is nucleic probe.Thus, it is possible to screen susceptible former efficiently
Send out the biological sample that the sex pili dyskinesia is levied.
Construct and reconstitution cell
According to the sixth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this structure
Build body and comprise the nucleic acid of foregoing separation.It should be noted that the nucleic acid of foregoing separation " construct comprise " table
Showing, the construct of the present invention comprises the nucleic acid of the DNAH5 gene mutation body compared with SEQ ID NO:1 with c.G8030A sudden change
Sequence, or comprise the nucleotide sequence of the DNAH11 gene mutation body compared with SEQ ID NO:3 with c.C6265T sudden change, or
Person comprises above-mentioned DNAH5 gene mutation body and the nucleotide sequence of DNAH11 gene mutation body simultaneously.Thus, the structure of the present invention is utilized
Build the reconstitution cell that body transformed acceptor cell obtains, it is possible to be efficiently used for the medicine that screening treatment primary ciliary dyskinesia is levied
Thing.Wherein, the kind of described recipient cell is not particularly limited, such as, can be Bacillus coli cells, mammalian cell, excellent
Select this receptor cell derived in mammal.
The term " construct " used in the present invention refers to such a kind of heredity carrier, and it comprises specific nucleic acid sequence
Row, and can purpose nucleotide sequence be proceeded in host cell, to obtain reconstitution cell.According to embodiments of the invention, structure
The form building body is not particularly limited.According to embodiments of the invention, it can be plasmid, phage, artificial chromosome, cosmid
(Cosmid), virus at least one, preferred plasmid.Plasmid, as heredity carrier, has simple to operate, can carry larger piece
The character of section, it is simple to operate and process.The form of plasmid is also not particularly limited, and both can be circular plasmids, it is also possible to be line
Character grain, can be i.e. strand, it is also possible to be double-strand.Those skilled in the art can select as required.At this
Term " nucleic acid " used in invention can be any polymer comprising deoxyribonucleotide or ribonucleotide, bag
Including but be not limited to through that modify or the most modified DNA, RNA, its length is the most any particular limitation.For being used for building
The construct of reconstitution cell, the most described nucleic acid is DNA, because DNA is for RNA, it is more stable, and is prone to behaviour
Make.
According to the seventh aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, should
Reconstitution cell is obtained by foregoing construct transformed acceptor cell.Thus, the reconstitution cell of the present invention can
At least one of DNAH5 gene mutation body entrained by effective expression construct and DNAH11 gene mutation body.According to the present invention
Some embodiments, utilize the reconstitution cell of the present invention, it is possible to the screening treatment medicine levied of primary ciliary dyskinesia effectively
Thing.According to embodiments of the invention, the kind of recipient cell is not particularly limited, such as, can be Bacillus coli cells, suckling
Zooblast, the most described recipient cell derives from non-human mammal.
It should be noted that in the method portion screening the biological sample that susceptible primary ciliary dyskinesia is levied herein above
Feature and advantage described in Fen, are equally applicable to screen the system of the biological sample that susceptible primary ciliary dyskinesia is levied
Or test kit, does not repeats them here.
In addition it is also necessary to explanation, the susceptible primary ciliary dyskinesia of screening according to embodiments of the present invention is levied
The method of biological sample, system and test kit, be that present inventor is through arduous creative work and Optimization Work
Just complete.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.If not specializing, the technological means employed in embodiment is this area
Conventional means known to technical staff, is referred to " Molecular Cloning: A Laboratory guide " third edition or Related product is carried out, institute
The reagent and the product that use also are available commercial.The various processes not described in detail and method are as known in the art
Conventional method, the source of agents useful for same, trade name and be necessary to list its constituent person, all indicate when occurring first,
Thereafter identical reagent used is if no special instructions, all identical with the content indicated first.
The order-checking of embodiment 1 full exon group determines Disease-causing gene and mutational site
1, sample collection:
Inventor collects 2 PCD patient's familys, is otherwise referred to as " PCD patient's family 1 " and " PCD patient in this article
Family 2 ".Wherein, Fig. 2 respectively illustrates the family collection of illustrative plates of above-mentioned 2 PCD patient's family PCD patient's familys 2.Wherein, Fig. 2 A is
The family collection of illustrative plates of PCD patient's family 1PCD patient's family 1, Fig. 2 B is the family collection of illustrative plates of PCD patient's family 2PCD patient's family 2.
As in figure 2 it is shown,Represent normal female；Expression cannot determine whether ill women；Represent normal male；Represent
Female patient；Represent late normal male；Represent late normal female；Represent male patient；Represent female
Property patient；Represent late and cannot determine whether ill women；Arrow indication is proband.As in figure 2 it is shown, PCD patient
Family 1, proband is young women, 29 years old；Show as upper respiratory tract checking the most repeatedly, sinusitis, bronchiectasis, the right side
Position heart and infertile.Proband has together younger sister blood younger sister, and because traffic accident is dead, it the most also has upper respiratory tract infection since the childhood, and a gas
Enlargement of pipe.Proband father and mother are consanguineous marriage, are normal person.In conjunction with proband's medical history, inspection and family history, proband is faced
Bed is diagnosed as PCD.
PCD patient's family 2, proband is female middle-aged, 36 years old, people from Hebei；Show as upper respiratory tract the most repeatedly to test
Card, sinusitis, bronchiectasis, dextrocardia are dirty.Proband father and mother are consanguineous marriage, are normal person；Proband gives birth to a female,
Healthy.In conjunction with proband person's medical history, inspection and the situation of father and mother's consanguineous marriage, proband is PCD by clinical diagnosis.
The pathological diagnosis of two proband is: primary ciliary dyskinesia is levied.
Inventor collects proband and the DNA sample of father and mother (normal person in family) thereof in the above-mentioned PCD patient's family 1 of acquisition
This, proband and the DNA sample of father and mother (normal person in family) thereof in PCD patient's family 2.
2, the order-checking of full exon group determines Disease-causing gene and mutational site
Inventor utilizes the NimbleGen SeqCap EZ Human Exome full exon trapping of Library v2.0 to put down
Platform, in conjunction with Illumina Hiseq 2000 high throughput sequencing technologies, to proband and father and mother thereof in above-mentioned 2 PCD patient's familys
(normal person in family) has all carried out the capture order-checking of full exon group, specifically comprises the following steps that
Prepared by 2.1 samples
Take proband and the peripheral blood of father and mother (normal person in family) thereof in above-mentioned 2 PCD patient's familys respectively, often utilize
Rule salting out method extracting genomic DNA, and utilize concentration and the purity of spectrophotometer measurement DNA, each specimen gene of gained
The OD260/OD280 of group DNA is respectively positioned between 1.7-2.0, and concentration is no less than 200ng/ μ l, and total amount is no less than 30 μ g, standby.
2.2 library constructions and order-checking
Ultrasonoscope (CovarisS2, Massachusetts, USA) is utilized to be broken at random by each genomic DNA sample
The fragment of 250-300 about bp, the operating instruction provided according to manufacturer subsequently, connect top connection respectively at fragment two ends
Preparation library (can be found in: the Illumina/Solexa standard that http://www.illumina.com/ provides builds storehouse description,
By referring to being incorporated by herein).Linear through Ligation-mediated PCR (LM-PCR) after library is purified
Amplification and capture agent SureSelect Biotiny lated RNA Library (BAITS) carry out hybridization enrichment, then pass through
The linear amplification of LM-PCR, be i.e. available on the machine after library detection is qualified order-checking, in order to obtains raw sequencing data.Wherein, reference
The cluster of Illumina standard and the protocol of order-checking check order, and order-checking platform is Illumina Hiseq 2000, reads
A length of 90bp, the average order-checking degree of depth of sample is 50 ×.
2.3 variation detections, annotation and data base compare
Utilize at the Illumina basecalling Software 1.7 raw sequencing data to above-mentioned acquisition
Reason, after filtering and depolluting, uses SOAPaligner/SOAP2 (to can be found in: Li R, Li Y, Kristiansen K, et
al,SOAP:short oligonucleotide alignment program.Bioinformatics 2008,24(5):
713-714；Li R,Yu C,Li Y,ea al,SOAP2:an improved ultrafast tool for short read
Alignment.Bioinformatics 2009,25 (15): 1966-1967, by referring to being incorporated by herein) comparison
To with reference to genome UCSC NCBI37/hg19, in order to obtain the comparison unique aligned sequences to genome.Then utilize
SOAPsnp (can be found in: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively
Parallel whole-genome resequencing.Genome Res 2009,19 (6): 1124-1132, by referring to inciting somebody to action
It is incorporated by herein) determine the genotype of target region.
After obtaining sequencing result, nonsynonymous mutation, acceptor splicing site/donor site sudden change, coding region are inserted and lacked
The sudden change losing this three class of suddenling change the most relevant to pathology is studied.As a result, inventor is found by sequencing analysis, for
In PCD patient's family 1, in the DNA sample of proband, find that 100289 single nucleotide polymorphism (SNPs) and 7059 are slotting
Enter/lack (Indels), the DNA sample of father proband finds 99191 SNPs and 6960 Indels, proband
The DNA sample of mother finds 97904 SNPs and 6981 Indels；For PCD patient's family 2, at the DNA sample of proband
100414 single nucleotide polymorphism (SNPs) and 7088 insertion/deletions (Indels) are found, father's proband in Ben
DNA sample finds 103106 SNPs and 7011 Indels, in the DNA sample of mother proband, finds 94530
SNPs and 7017 Indels.Subsequently by dbSNP data base (http://hgdownload.cse.ucsc.edu/
GoldenPath/hg19/database/snp132.txt.gz), HapMap data base (ftp: //
Ftp.ncbi.nlm.nih.gov/hapmap), thousand human genome data bases (ftp: //
Ftp.1000genomes.ebi.ac.uk/vol1/ftp), Yan Di and Huang Di, two legendary rulers of remote antiquity data base (http://yh.genomics.org.cn/) etc.
The filtration of public database, removes all known and (variation MAF value is high, is usually the most not in the high variation of data base's medium frequency
The common polymorphism caused a disease).Filter out intron or the sudden change of intergenic region and the samesense mutation in other regions, i.e. mistake simultaneously
Filter the sudden change not affecting protein structure and function.Then, inventor combines practical situation and the PCD feature of two PCD familys,
The sudden change utilizing recessive inheritance's Policy Filtering to obtain is further analyzed.Then, will analyze after result with reported at present
The list of genes relevant to PCD in road takes common factor.As a result, inventor obtains DNAH5 gene from PCD patient's family 1
C.G8030A suddenlys change (p.R2677Q), obtains the c.C6265T sudden change of DNAH11 gene from PCD patient's family 2
(p.R2089*)。
Analyzing further and understand, in PCD patient's family 1, proband has the homozygous mutation of c.G8030A, its normal father and mother
Being heterozygous mutant in this site, this sports the new mutation of known and meets normal hidden model, considers that sequencing quality is high simultaneously
And SIFT prediction sudden change has hazard factor, inventor it was initially believed that, and this sports the pathogenic mutation of this family；PCD patient's family 2
The homozygous mutation of middle proband c.C6265T, its normal father and mother are heterozygous mutant in this site, and this sports the new of known
Suddenly change and meet normal hidden model, considering that sequencing quality is high and SIFT prediction sudden change has hazard factor, inventor it was initially believed that simultaneously
This sports the pathogenic mutation of this family.
Owing to the order-checking of exon group exists a certain degree of false positive, it follows that inventor utilizes again Sanger order-checking side
Above-mentioned 2 sudden changes determined are verified in the proband of 2 PCD patient's familys by method respectively, result display DNAH5
The c.G8030A sudden change of gene and the c.C6265T sudden change of DNAH11 gene are real sudden change.Thus, show DNAH5 and
DNAH11 gene is the pathogenic related gene that primary ciliary dyskinesia is levied, c.G8030A (DNAH5) and c.C6265T
(DNAH11) sudden change is the pathogenic mutation that primary ciliary dyskinesia is levied.
The pathogenic mutation that embodiment 2 Sanger method sequence verification primary ciliary dyskinesia is levied
Respectively to 4 in 3 people (proband and the father and mother acted normally thereof) in PCD patient's family 1, PCD patient's family 2
DNAH5 and the DNAH11 gene of people (proband and for the father and mother that act normally) (see Fig. 2) detects, for DNAH5 base
The c.G8030A mutational site of cause and place, the c.C6265T mutational site primers of DNAH11 gene, then pass through
The method of PCR amplification, product purification and order-checking obtains the relevant sequence of said mutation, according to determining that sequencing results belongs to prominent
Modification or wild type, be heterozygous mutant or homozygous mutation, and sequence and phenotype in family whether in being divided into from testing
Card DNAH5 and DNAH11 and primary ciliary dyskinesia levy between dependency.Concrete grammar step is as follows:
1, DNA extraction
Gather respectively in above-mentioned PCD patient's family 1 and proband and the father and mother acted normally, PCD patient's family 2 are first demonstrate,proved
Person and the father and mother acted normally thereof, then utilize QIAmp Blood kit (Qiagen, Hilden, Germany) to extract peripheral blood
Genomic DNA in leukocyte, and utilize Qubit Fluorometer and agarose gel electrophoresis to measure the dense of extracted DNA
Degree and purity, the OD of each specimen genomic DNA of gained260/OD280Being respectively positioned between 1.7-2.0, concentration is no less than 50ng/
Microlitre, total amount is no less than 3 μ g, thus, it is thus achieved that each DNA sample, standby.
2, design of primers and PCR reaction
First, reference man genoid data unit sequence storehouse GRCh37.1/hg19, design obtains having SEQ ID NO:5-6
The DNAH5 gene extron specific primer of shown nucleotide sequence, and there is the nucleotide shown in SEQ ID NO:7-8
The DNAH11 gene extron specific primer of sequence, is specifically shown in following table.
DNAH5 and DNAH11 gene extron specific primer
Then, prepare the PCR reaction system of each genomic DNA sample according to following proportioning respectively and carry out PCR reaction.
Outside for the c.G8030A mutational site of DNAH5 gene and the place, c.C6265T mutational site of DNAH11 gene
Aobvious subsequence, prepares each PCR reaction system of each genomic DNA sample the most respectively:
Reaction system: 25 μ l
Then, (different is prominent according to following reaction condition, the preparation each PCR reaction system of acquisition to be carried out PCR reaction respectively
Displacement point uses identical reaction condition):
Reaction condition:
Thus, it is thus achieved that in above-mentioned PCD patient's family 1 in proband and the father and mother acted normally, PCD patient's family 2 first
Card person and the pcr amplification product of father and mother acted normally thereof.
3, order-checking
By in step 2 obtain available from proband in PCD patient's family 1 and the father and mother acted normally, PCD patient's family
In 2, proband and father and mother's pcr amplification product of acting normally thereof, carry out DNA sequencing.Wherein, order-checking uses the order-checking of ABI3730 type
Instrument
Based on sequencing result, above-mentioned each sample is carried out DNAH5 and DNAH11 gene coded sequence comparison.Invention Crinis Carbonisatus
Existing, in patient (proband) in two familys and family thereof normal person (in PCD patient's family 1, the father and mother of proband, PCD suffer from
The father and mother of proband in person's family 2) at c.G8030A (DNAH5) and c.C6265T (DNAH11), segregation phenomenon altogether occurs respectively,
The patient of PCD patient's family 1 and PCD patient's family 2 carries c.G8030A and c.C6265T sudden change respectively.Thus, it was demonstrated that DNAH5
The c.G8030A sudden change of gene, the c.C6265T sudden change of DNAH11 gene are the pathogenic mutations that primary ciliary dyskinesia is levied.
Wherein, proband in Fig. 3 shows PCD patient's family 1 and the DNAH5 gene mutation site of father and mother thereof
Sanger sequence verification peak figure, Fig. 4 shows the DNAH11 gene mutation position of the proband in PCD patient's family 2 and father and mother thereof
The Sanger sequence verification peak figure of point.From Fig. 3 and 4, in PCD patient's family 1, the DNAH5 gene of patient has c.G8030A
Sudden change, and the normal person in this family does not carry this sudden change；In PCD patient's family 2, the DNAH11 gene of patient has
C.C6265T suddenlys change, and the normal person in this family does not carry this sudden change.Thus, DNAH5 gene is proved further
C.G8030A sudden change, the c.C6265T sudden change of DNAH11 gene are the pathogenic mutations that primary ciliary dyskinesia is levied.
It should be noted that DNAH5 is known PCD Disease-causing gene, both at home and abroad the pathogenic of this gene is carried out in a large number
Research, DNAH5 accounts for the 28% of total PCD Disease-causing gene, and is considered as the principal causative gene of PCD.The pathogenesis basis of PCD
The textural anomaly or the regulation and control power cilium process appearance that are power cilium are abnormal.Power ciliary structures is 9+2 structure, and central authorities are two
Individual center microtubule, is around formed 9 made periphery microtubule, dynamic arm, chain T-Ring and spoke by secondary fiber A and time fiber B
Be connected mutually.Power arm has two, is inner side power arm (IDA) and outside power arm (ODA) respectively, at power ciliary movement
In play a crucial role.Major part research at present is thought, DNAH5 sudden change can cause outside dynein arm textural anomaly, but also has
It is the most abnormal that research thinks that DNAH5 sudden change can also result in interior outside power arm.DNAH11 is also the pathogenic base of known PCD
Cause, this gene is pathogenic the clearest and the most definite at present.DNAH11 is positioned at No. 7 the short arm of a chromosome, has 82 exons.Research is aobvious at present
Showing, DNAH11 does not cause the textural anomaly of power cilium, and its pathogenic characteristic is mainly by the mediation system pendulum to power cilium
The raw impact of movable property.
To sum up, inventor further demonstrate DNAH5 and DNAH11 gene be primary ciliary dyskinesia levy cause a disease
Related gene, the c.G8030A sudden change of DNAH5 gene, the c.C6265T sudden change of DNAH11 gene are primary ciliary dyskinesias
The pathogenic mutation levied.
Embodiment 3 detection kit
Preparing a detection kit, it comprises at least one draw being adapted to detect for DNAH5 and DNAH11 gene mutation body
(wherein compared with SEQ ID NO:1, described DNAH5 gene mutation body has c.G8030A sudden change to thing；With SEQ ID NO:3 phase
Ratio, described DNAH11 gene mutation body has c.C6265T sudden change), in order to it is susceptible to suffer from primary ciliary dyskinesia for screening and levies
Biological sample, wherein these primers include that the DNAH5 gene extron specific primer shown in SEQ ID NO:5-6 is (see reality
Execute example 1), and the DNAH11 gene extron specific primer (see embodiment 1) shown in SEQ ID NO:7-8.
Concretely comprising the following steps of utilize the screening of mentioned reagent box to be susceptible to suffer from biological sample that primary ciliary dyskinesia levies: according to
Method described in the step 1 of embodiment 2 extracts person DNA to be measured, draws with above-mentioned exon specificity with the DNA extracted for template
Thing carries out PCR reaction, and according to this area conventional method to PCR primer purification, is checked order by the product of purification, then pass through
Observe whether the sequence obtained by order-checking has at least one sudden change selected from c.G8030A and c.C6265T, thus effectively examine
Survey person to be measured whether to be susceptible to suffer from primary ciliary dyskinesia and levy, further, it is possible to from person to be measured, filter out that to be susceptible to suffer from constitutional fine
The biological sample that the hair dyskinesia is levied.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
Claims (10)
1. the nucleic acid separated, it is characterised in that
Compared with SEQ ID NO:3, described nucleic acid has c.C6265T sudden change,
Optionally, described nucleic acid is DNA.
2. the polypeptide separated, it is characterised in that
Compared with SEQ ID NO:4, the polypeptide of described separation has p.R2089* sudden change,
Optionally, described polypeptide is by the nucleic acid coding described in claim 1.
3. the method screening the biological sample that susceptible primary ciliary dyskinesia is levied, it is characterised in that include following step
Rapid:
From described extraction from biological material sample of nucleic acid；
Determine the nucleotide sequence of described sample of nucleic acid；
The nucleotide sequence of described sample of nucleic acid or its complementary series, have c.C6265T sudden change compared with SEQ ID NO:3, be institute
State the instruction that the susceptible primary ciliary dyskinesia of biological sample is levied,
Optionally, described biological sample is selected from least one of blood of human body, skin, hair and muscle,
Optionally, described sample of nucleic acid is complete genome DNA.
Method the most according to claim 3, it is characterised in that wrap further from described extraction from biological material sample of nucleic acid
Include:
From described extraction from biological material RNA sample, the most described RNA sample is mRNA；And
Based on described RNA sample, passing through reverse transcription reaction, it is thus achieved that cDNA sample, described cDNA sample constitutes described sample of nucleic acid.
Method the most according to claim 3, it is characterised in that determine that the nucleotide sequence of described sample of nucleic acid wraps further
Include:
For described sample of nucleic acid, build nucleic acid sequencing library；And
Is checked order in described nucleic acid sequencing library, in order to obtain the sequencing result being made up of multiple sequencing datas,
Optionally, use at least one selected from Hiseq2000, SOLiD, 454 and single-molecule sequencing device that described nucleic acid is surveyed
Check order in preface storehouse,
Optionally, for described sample of nucleic acid, build nucleic acid sequencing library and farther include:
Utilize at least one being selected from DNAH11 gene extron specific primer, described sample of nucleic acid is carried out PCR amplification；With
And
For obtained amplified production, build described nucleic acid sequencing library,
Optionally, described DNAH11 gene extron specific primer has the nucleotide sequence as shown in SEQ ID NO:7-8.
6. the system screening the biological sample that susceptible primary ciliary dyskinesia is levied, it is characterised in that including:
Nucleic acid-extracting apparatus, described nucleic acid-extracting apparatus is for from described extraction from biological material sample of nucleic acid；
Nucleotide sequence determines that device, described nucleotide sequence determine that device is connected with described nucleic acid-extracting apparatus, for described core
Acid sample is analyzed, in order to determine the nucleotide sequence of described sample of nucleic acid；
Judgment means, with described nucleotide sequence, described judgment means determines that device is connected, in order to core based on described sample of nucleic acid
Acid sequence or its complementary series, have c.C6265T sudden change, it is judged that described biological sample is the most susceptible compared with SEQ ID NO:3
Primary ciliary dyskinesia is levied,
Optionally, described nucleic acid-extracting apparatus farther includes:
RNA extraction unit, described RNA extraction unit is for from described extraction from biological material RNA sample；And
Reverse transcription unit, described reverse transcription unit is connected with described RNA extraction unit, for inverting described RNA sample
Record reaction, in order to obtain cDNA sample, described cDNA sample constitutes described sample of nucleic acid.
System the most according to claim 6, it is characterised in that described nucleotide sequence determines that device farther includes:
Library construction unit, described library construction unit, for for described sample of nucleic acid, builds nucleic acid sequencing library；And
Order-checking unit, described order-checking unit is connected with described library construction unit, for surveying described nucleic acid sequencing library
Sequence, in order to obtain the sequencing result being made up of multiple sequencing datas,
Optionally, described library construction unit farther includes:
PCR expands module, is provided with DNAH11 gene extron specific primer at least one in described PCR amplification module,
To utilize at least one of DNAH11 gene extron specific primer, described sample of nucleic acid is carried out PCR amplification,
Optionally, described DNAH11 gene extron specific primer has the nucleotide sequence as shown in SEQ ID NO:7-8,
Optionally, described order-checking unit includes selected from least one of HISEQ2000, SOLiD, 454 and single-molecule sequencing device.
8. the test kit being used for screening the biological sample that susceptible primary ciliary dyskinesia is levied, it is characterised in that contain:
It is adapted to detect for the reagent of at least one of DNAH11 gene mutation body, wherein
Compared with SEQ ID NO:3, described DNAH11 gene mutation body has c.C6265T sudden change,
Optionally, described reagent is nucleic probe or primer.
9. a construct, it is characterised in that comprise the nucleic acid of separation described in claim 1.
10. a reconstitution cell, it is characterised in that described reconstitution cell is to be converted by the construct described in claim 9 to be subject to
Somatic cell and obtain.
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| CN201610665577.3A CN106282195A (en) | 2013-04-28 | 2013-04-28 | Gene mutation body and application thereof |
| HK17104940.4A HK1231503A1 (en) | 2013-04-28 | 2017-05-17 | Gene mutant and application thereof |
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| CN201610665577.3A CN106282195A (en) | 2013-04-28 | 2013-04-28 | Gene mutation body and application thereof |
| CN201310157540.6A CN104120133A (en) | 2013-04-28 | 2013-04-28 | Gene mutant and application thereof |
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| CN201610665577.3A Pending CN106282195A (en) | 2013-04-28 | 2013-04-28 | Gene mutation body and application thereof |
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Cited By (4)
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| CN110438220A (en) * | 2019-08-16 | 2019-11-12 | 深圳市人民医院 | The motionless syndrome gene panel kit of cilium and its application |
| CN110551728A (en) * | 2018-06-04 | 2019-12-10 | 中国人民解放军总医院 | FOXC1 gene mutant and application thereof |
| CN114540384A (en) * | 2020-12-31 | 2022-05-27 | 中山大学附属第一医院 | Oligonucleotides for reducing angiotensin converting enzyme 2(ACE2) expression and their use in treating viral infections |
| CN116804219A (en) * | 2023-06-13 | 2023-09-26 | 广州中景医学检验实验室有限公司 | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation |
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| AU2017271665B2 (en) | 2016-05-27 | 2023-11-23 | Transcriptx, Inc. | Treatment of primary ciliary dyskinesia with synthetic messenger RNA |
| CN113584042A (en) * | 2021-07-20 | 2021-11-02 | 上海市儿童医院 | ODNAH gene new mutation and application thereof in severe hypospadias detection |
| CN113584152A (en) * | 2021-07-20 | 2021-11-02 | 上海市儿童医院 | Application of ODNAH gene mutation in severe hypospadias detection |
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| CN103205503B (en) * | 2010-12-28 | 2014-12-24 | 深圳华大基因科技有限公司 | Method for detecting CLCN1 gene mutation, kit and specific primer |
| CN102409089B (en) * | 2011-10-08 | 2017-04-19 | 深圳华大基因股份有限公司 | Kit, method and application for detecting mutation of predetermined locus in DNA sample |
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2013
- 2013-04-28 CN CN201310157540.6A patent/CN104120133A/en active Pending
- 2013-04-28 CN CN201610665577.3A patent/CN106282195A/en active Pending
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- 2017-05-17 HK HK17104940.4A patent/HK1231503A1/en unknown
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| BARTOLONI L, ET AL.: "Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia", 《PNAS》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551728A (en) * | 2018-06-04 | 2019-12-10 | 中国人民解放军总医院 | FOXC1 gene mutant and application thereof |
| CN110551728B (en) * | 2018-06-04 | 2021-07-20 | 中国人民解放军总医院 | FOXC1 gene mutant and application thereof |
| CN110438220A (en) * | 2019-08-16 | 2019-11-12 | 深圳市人民医院 | The motionless syndrome gene panel kit of cilium and its application |
| CN114540384A (en) * | 2020-12-31 | 2022-05-27 | 中山大学附属第一医院 | Oligonucleotides for reducing angiotensin converting enzyme 2(ACE2) expression and their use in treating viral infections |
| CN116804219A (en) * | 2023-06-13 | 2023-09-26 | 广州中景医学检验实验室有限公司 | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104120133A (en) | 2014-10-29 |
| HK1231503A1 (en) | 2017-12-22 |
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