CN103509802A - SLC25A13 gene mutant and its application - Google Patents
SLC25A13 gene mutant and its application Download PDFInfo
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- CN103509802A CN103509802A CN201210205478.9A CN201210205478A CN103509802A CN 103509802 A CN103509802 A CN 103509802A CN 201210205478 A CN201210205478 A CN 201210205478A CN 103509802 A CN103509802 A CN 103509802A
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Abstract
The invention relates to separated nucleic acid of encoded SLC25A13 mutants, separated polypeptide, a method for screening an NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) disease susceptible biological sample, a system for screening an NICCD disease susceptible biological sample and a kit for screening an NICCD disease susceptible biological sample. Specifically, the separated nucleic acid of encoded SLC25A13 mutants, compared with the SEQ ID NO:1, has at least one mutation selected from the following: c.754G>A and c.1177+1G>A. By detecting the existence of the new mutants in biological samples, whether the biological samples are susceptible to the NICCD disease can be effectively detected.
Description
Technical field
The present invention relates to SLC25A13 gene mutation body and application thereof.Particularly, the present invention relates to nucleic acid, the isolated polypeptide of separated coding SLC25A13 mutant, the method for the biological sample of screening susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases, the system of the biological sample of screening susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases and for screening the test kit of the biological sample of susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases.
Background technology
Newborn infant's melon amino acid mass formed by blood stasis 2 type (phaeohyphomycosis, be for No. OMIM 605814), be again intrahepatic cholestasis (the Neonatal Intrahepatic Cho lestasis Caused By Ctrin Deficiency of newborn infant's vitamin P due to lacking, NICCD), it belongs to a kind of of melon amino acid metabolic disease in Aminoacidopathies, in birth, between to five months, can there is ChJ in patient, liver function is undesired, there is multiple high Amino acid mass formed by blood stasis, the symptoms such as galactosemia and fatty liver.Severe patient can cause growth retardation, the more than or anaemia of abnormal bleeding, hypoglycemia, liver enlargement, even liver failure.The incidence of this disease estimates according to gene mutation frequency, and Taiwan incidence is about 1/14000.NICCD is a kind of autosomal recessive hereditary diseases, and SLC25A13 gene is current unique known gene relevant with NICCD.Up to now, on SLC25A13 gene, find a plurality of mutational sites and NICCD disease-related, but also had a plurality of NICCD patients not find sudden change.
Thereby, at present the research of NICCD disease is still needed deeply.
Summary of the invention
The present invention is intended at least solve one of technical problem existing in prior art.For this reason, one object of the present invention is to propose the new mutant on the Disease-causing gene of newborn infant's melon amino acid mass formed by blood stasis 2 type diseases (in this article sometimes also referred to as " NICCD disease ").
The following work of the present invention based on contriver completes: the method that contriver catches order-checking by target area has been determined the new mutant on the Disease-causing gene of NICCD disease.
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding SLC25A13 mutant of separation.According to embodiments of the invention, compare with SEQ ID NO:1, this nucleic acid has and is selected from least one following sudden change: c.754G > A and c.1177+1G > A.According to embodiments of the invention, contriver has determined the new mutant body of a plurality of SLC25A13 genes, the morbidity of these new mutant bodies and NICCD disease is closely related, thereby by detecting these new mutant bodies, in biological sample, whether exist, effectively detection of biological sample susceptible NICCD disease whether.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, to compare with SEQID NO:2, this isolated polypeptide has and is selected from least one following sudden change: p.Glu252Lys and p.Gly393Glyfs*2.According to concrete examples more of the present invention, this polypeptide is by the nucleic acid encoding of the coding SLC25A13 mutant of aforementioned separation.By whether expressing this polypeptide in detection of biological sample, detection of biological sample susceptible NICCD disease whether effectively.
According to a third aspect of the invention we, the present invention proposes a kind of method of screening the biological sample of susceptible NICCD disease.According to embodiments of the invention, the method for the biological sample of this screening susceptible NICCD disease comprises the following steps: from extraction from biological material sample of nucleic acid; The nucleotide sequence of definite kernel acid sample; The nucleotide sequence of this sample of nucleic acid or its complementary sequence are compared with SEQ ID NO:1, have and are selected from c.754G > A and c.1177+1G > at least one sudden change of A is the indication of biological sample susceptible NICCD disease.By according to the method for the biological sample of the screening susceptible NICCD disease of the embodiment of the present invention, can effectively screen the biological sample of susceptible NICCD disease.
According to a forth aspect of the invention, the present invention proposes a kind of system of screening the biological sample of susceptible NICCD disease.According to embodiments of the invention, the system of the biological sample of this screening susceptible NICCD disease comprises: nucleic acid-extracting apparatus, and this nucleic acid-extracting apparatus is used for from extraction from biological material sample of nucleic acid; Nucleotide sequence determining device, this nucleotide sequence determining device is connected with nucleic acid-extracting apparatus, for sample of nucleic acid is analyzed, so that the nucleotide sequence of definite kernel acid sample; Judgment means, this judgment means is connected with nucleotide sequence determining device, so that the nucleotide sequence based on sample of nucleic acid or its complementary sequence are compared with SEQ ID NO:1, whether have and be selected from c.754G A and c.1177+1G > at least one sudden change of A, judge whether susceptible NICCD disease of biological sample.Utilize this system, can effectively implement the method for the biological sample of aforementioned screening susceptible NICCD disease, thereby can effectively screen the biological sample of susceptible NICCD disease.
According to a fifth aspect of the invention, the present invention proposes a kind of for screening the test kit of the biological sample of susceptible NICCD disease.According to embodiments of the invention, should comprise for screening the test kit of the biological sample of susceptible NICCD disease: the reagent that is suitable for detecting SLC25A13 gene mutation body, wherein compare with SEQ ID NO:1, SLC25A13 gene mutation body have be selected from c.754G > A and c.1177+1G > at least one sudden change of A.Utilize test kit according to an embodiment of the invention, can effectively screen the biological sample of susceptible NICCD disease.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage accompanying drawing below combination obviously and is easily understood becoming the description of embodiment, wherein:
Fig. 1: shown according to the system of biological sample and the schematic diagram of integral part thereof of the screening susceptible NICCD disease of the embodiment of the present invention, wherein, A is according to the schematic diagram of the system of the biological sample of the screening susceptible NICCD disease of the embodiment of the present invention, B is according to the schematic diagram of the nucleic acid-extracting apparatus of the embodiment of the present invention, and C is according to the schematic diagram of the nucleotide sequence determining device of the embodiment of the present invention;
Fig. 2: shown according to the embodiment of the present invention to patient and father and mother and uncle's SLC25A13 gene mutation site c.1177+1G > the Sanger sequence verification peak figure of A; And
Fig. 3: shown according to the embodiment of the present invention to patient and father and mother and uncle's SLC25A13 gene mutation site c.754G > the Sanger sequence verification peak figure of A.
Embodiment
Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Below by the embodiment being described with reference to the drawings, be exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
SLC25A13 gene mutation body
According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding SLC25A13 mutant of separation.According to embodiments of the invention, compare with SEQ ID NO:1, this nucleic acid has and is selected from least one following sudden change: c.754G > A and c.1177+1G > A, preferably, c.754G this nucleic acid have simultaneously > A and c.1177+1G > A sudden change.The phraseology used in this article " nucleic acid of coding SLC25A13 mutant ", refer to the nucleic acid substances corresponding with the gene of the SLC25A13 mutant of encoding, the type that is nucleic acid is not particularly limited, can be any deoxyribonucleotide corresponding with the encoding gene of SLC25A13 mutant and/or polymkeric substance of ribonucleotide of comprising, include but not limited to DNA, RNA or cDNA.According to a concrete example of the present invention, the nucleic acid of foregoing coding SLC25A13 mutant is DNA.According to embodiments of the invention, contriver the is true new mutant body of a plurality of SLC25A13 genes, the morbidity of these new mutant bodies and NICCD disease is closely related, thereby whether exist in biological sample by detecting these new mutant bodies, detection of biological sample susceptible NICCD disease whether effectively, also can in organism, whether exist by detecting these mutant, can effectively predict whether susceptible NICCD disease of organism.
The nucleic acid of these codings SLC25A13 mutant, is that present inventor catches the method for order-checking by target area, for clinical samples, checks order, and the new mutant on the Disease-causing gene of definite NICCD disease.
It should be noted that, the cDNA sequence of SLC25A13 gene is as follows:
ATGGCGGCCGCCAAGGTGGCTTTAACCAAGAGAGCAGATCCAGCTGAGCTTAGAACAATATTTTTGAAGTATGCAAGCATTGAGAAAAACGGTGAATTTTTCATGTCCCCCAATGACTTTGTCACTCGATACTTGAACATTTTTGGAGAAAGCCAGCCTAATCCAAAGACTGTGGAACTTTTAAGTGGAGTGGTGGATCAGACCAAAGATGGATTAATATCTTTTCAAGAATTTGTTGCCTTTGAATCTGTCCTGTGTGCCCCTGATGCTTTGTTTATGGTAGCCTTTCAGCTGTTTGACAAAGCTGGCAAAGGAGAAGTAACTTTTGAGGATGTTAAGCAAGTTTTTGGACAGACCACAATTCATCAACATATTCCATTTAACTGGGATTCAGAATTTGTGCAACTACATTTTGGAAAAGAAAGAAAAAGACACCTGACATATGCGGAATTTACTCAGTTTTTATTGGAAATACAACTGGAGCACGCAAAGCAAGCCTTTGTGCAACGGGACAATGCTAGGACTGGGAGAGTCACAGCCATCGACTTCCGAGACATCATGGTCACCATCCGCCCCCATGTCTTGACTCCTTTTGTAGAAGAATGTCTAGTAGCTGCTGCTGGAGGTACCACATCCCATCAAGTTAGTTTCTCCTATTTTAATGGATTTAATTCGCTCCTTAACAACATGGAACTCATTAGAAAGATCTATAGCACTCTGGCTGGCACCAGGAAAGATGTTGAAGTGACTAAGGAGGAGTTTGTTCTGGCAGCTCAGAAATTTGGTCAGGTTACACCCATGGAAGTTGACATCTTGTTTCAGTTAGCAGATTTATATGAGCCAAGGGGACGTATGACCTTAGCAGACATTGAACGGATTGCTCCTCTGGAAGAGGGAACTCTGCCCTTTAACTTGGCTGAGGCCCAGAGGCAGAAGGCCTCAGGTGATTCAGCTCGACCAGTTCTTCTACAAGTTGCAGAGTCGGCCTACAGGTTTGGTCTGGGTTCTGTTGCTGGAGCTGTTGGAGCCACTGCTGTGTATCCTATCGATCTTGTAAAAACTCGAATGCAGAACCAACGATCAACTGGCTCTTTTGTGGGAGAACTCATGTATAAAAACAGCTTTGACTGTTTTAAGAAAGTGCTACGCTATGAAGGCTTCTTTGGACTGTATAGAGGTCTGTTGCCACAGTTATTGGGAGTTGCCCCAGAGAAGGCCATAAAACTTACAGTGAACGATTTTGTGAGGGATAAATTTATGCACAAAGATGGTTCGGTCCCACTTGCAGCAGAAATTCTTGCTGGAGGCTGCGCTGGAGGCTCCCAGGTGATTTTCACAAATCCTTTAGAAATCGTCAAGATCCGTTTGCAAGTGGCAGGAGAAATCACCACTGGTCCTCGAGTCAGTGCTCTGTCTGTCGTGCGGGACCTGGGGTTTTTTGGGATCTACAAGGGTGCCAAAGCATGCTTTCTGCGGGACATTCCTTTCTCGGCCATCTACTTTCCGTGCTATGCTCATGTGAAGGCTTCCTTTGCAAATGAAGATGGGCAGGTTAGCCCAGGAAGCCTGCTCTTAGCTGGTGCCATAGCTGGTATGCCTGCAGCATCTTTAGTGACCCCTGCTGATGTTATCAAGACGAGATTACAGGTGGCTGCCCGGGCTGGCCAAACCACTTACAGCGGAGTGATAGACTGCTTTAGAAAGATACTGCGTGAAGAAGGACCAAAAGCTCTGTGGAAGGGAGCTGGTGCTCGTGTATTTCGATCCTCACCCCAGTTTGGTGTAACTTTGCTGACTTACGAATTGCTACAGCGATGGTTCTACATTGATTTTGGAGGAGTAAAACCCATGGGATCAGAGCCAGTTCCTAAATCCAGGATCAACCTGCCTGCCCCGAATCCTGATCACGTTGGGGGCTACAAACTGGCAGTTGCTACATTTGCAGGGATTGAAAACAAATTTGGACTTTACCTACCTCTCTTCAAGCCATCAGTATCTACCTCAAAGGCTATTGGTGGAGGCCCATAG（SEQ?ID?NO：1），
The aminoacid sequence of its coding is as follows:
MAAAKVALTKRADPAELRTIFLKYASIEKNGEFFMSPNDFVTRYLNIFGESQPNPKTVELLSGVVDQTKDGLISFQEFVAFESVLCAPDALFMVAFQLFDKAGKGEVTFEDVKQVFGQTTIHQHIPFNWDSEFVQLHFGKERKRHLTYAEFTQFLLEIQLEHAKQAFVQRDNARTGRVTAIDFRDIMVTIRPHVLTPFVEECLVAAAGGTTSHQVSFSYFNGFNSLLNNMELIRKIYSTLAGTRKDVEVTKEEFVLAAQKFGQVTPMEVDILFQLADLYEPRGRMTLADIERIAPLEEGTLPFNLAEAQRQKASGDSARPVLLQVAESAYRFGLGSVAGAVGATAVYPIDLVKTRMQNQRSTGSFVGELMYKNSFDCFKKVLRYEGFFGLYRGLLPQLLGVAPEKAIKLTVNDFVRDKFMHKDGSVPLAAEILAGGCAGGSQVIFTNPLEIVKIRLQVAGEITTGPRVSALSVVRDLGFFGIYKGAKACFLRDIPFSAIYFPCYAHVKASFANEDGQVSPGSLLLAGAIAGMPAASLVTPADVIKTRLQVAARAGQTTYSGVIDCFRKILREEGPKALWKGAGARVFRSSPQFGVTLLTYELLQRWFYIDFGGVKPMGSEPVPKSRINLPAPNPDHVGGYKLAVATFAGIENKFGLYLPLFKPSVSTSKAIGGGP（SEQ?ID?NO：2）。
SLC25A13 gene is the Disease-causing gene of NICCD disease.For this reason, contriver carries out target area for NICCD Disease and catches order-checking, thereby has determined a plurality of SLC25A13 gene mutation bodies, c.754G > A and c.1177+1G > A, the morbidity height correlation of these mutant and NICCD disease.
Further, contriver finds, when occur c.754G in SLC25A13 gene mutation body simultaneously > A and c.1177+1G > A is when suddenly change, and the susceptibility of NICCD disease is higher.By detecting these SLC25A13 gene mutation bodies with a plurality of mutational sites, in biological sample, whether exist, can be further detection of biological sample susceptible NICCD disease whether effectively, also can in organism, whether exist by detecting these mutant, can effectively predict whether susceptible NICCD disease of organism.
According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compare with SEQ ID NO:2, this isolated polypeptide has and is selected from least one following sudden change: p.Glu252Lys and p.Gly393Glyfs*2, preferably, this polypeptide has p.Glu252Lys and p.Gly393Glyfs*2 sudden change simultaneously.According to concrete examples more of the present invention, this polypeptide is by the nucleic acid encoding of the coding SLC25A13 mutant of aforementioned separation.Wherein, it should be noted that, " p.Gly393Glyfs*2 " sudden change forms new spliceosome, therefore compare with SEQ ID NO:2, the 393rd amino acids of this isolated polypeptide is sudden change not, and stops after the 394th amino acids, and new protein has 394 amino acid.By whether expressing this polypeptide in detection of biological sample, whether detection of biological sample susceptible NICCD disease whether, also can exist by detecting these polypeptide in organism effectively, can effectively predict whether susceptible NICCD disease of organism.
The method of the biological sample of screening susceptible NICCD disease
According to a third aspect of the invention we, the present invention proposes a kind of method of screening the biological sample of susceptible NICCD disease.According to embodiments of the invention, the method for the biological sample of this screening susceptible NICCD disease can comprise the following steps:
First, from extraction from biological material sample of nucleic acid.According to embodiments of the invention, the type of biological sample is also not particularly limited, as long as can extract the sample of nucleic acid whether reflection biological sample SLC25A13 gene exists sudden change from this biological sample.According to embodiments of the invention, biological sample can be any tissue of human body.According to concrete examples more of the present invention, preferably, biological sample is to be selected from least one of blood of human body, skin, hair and muscle.Thus, can sample easily and detect, thereby can further improve the efficiency of the biological sample of screening susceptible NICCD disease.According to embodiments of the invention, here the term that used " sample of nucleic acid " should be interpreted broadly, it can be anyly can reflect whether SLC25A13 gene in biological sample exists the sample of sudden change, it can be for example the complete genome DNA directly extracting from biological sample, also can be a part that comprises SLC25A13 gene coded sequence in this full genome, can be the total RNA extracting from biological sample, can be also the mRNA extracting from biological sample.According to one embodiment of present invention, described sample of nucleic acid is complete genome DNA.Thus, can expand the source range that comes of biological sample, and can to the much information of biological sample, determine simultaneously, thereby can improve the efficiency of the biological sample of screening susceptible NICCD disease.In addition, according to embodiments of the invention, for adopting RNA as sample of nucleic acid, from extraction from biological material sample of nucleic acid, may further include: from extraction from biological material RNA sample, preferably RNA sample is mRNA; And based on resulting RNA sample, by reverse transcription reaction, obtain cDNA sample, resulting cDNA composition of sample sample of nucleic acid.Thus, can further improve and utilize RNA as the efficiency of the biological sample of sample of nucleic acid screening susceptible NICCD disease.
Next, after obtaining sample of nucleic acid, can analyze sample of nucleic acid, thereby can determine the nucleotide sequence of resulting sample of nucleic acid.According to embodiments of the invention, determine resulting sample of nucleic acid nucleotide sequence method and apparatus and be not particularly limited.According to a particular embodiment of the invention, can pass through sequence measurement, the nucleotide sequence of definite kernel acid sample.According to embodiments of the invention, can and be not particularly limited for the method and apparatus that checks order.According to embodiments of the invention, can adopt s-generation sequencing technologies, also can adopt the third generation and the 4th generation or more advanced sequencing technologies.According to concrete example of the present invention, can utilize be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device nucleotide sequence is checked order.Thus, in conjunction with up-to-date sequencing technologies, for Single locus, can reach the higher order-checking degree of depth, detection sensitivity and accuracy improve greatly, thereby can utilize the high-throughput of these sequencing devices, the feature of degree of depth order-checking, further improve sample of nucleic acid is detected to the efficiency of analyzing.Thereby, can improve follow-up accuracy and accuracy when sequencing data is analyzed.Thus, according to embodiments of the invention, the nucleotide sequence of definite kernel acid sample may further include: first, for resulting sample of nucleic acid, build nucleic acid sequencing library; And checked order in resulting nucleic acid sequencing library, to obtain the sequencing result being formed by a plurality of sequencing datas.According to some embodiments of the present invention, can adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in resulting nucleic acid sequencing library.In addition, according to embodiments of the invention, can screen sample of nucleic acid, enrichment target area sequence be SLC25A13 gene c.754G > A and c.1177+1G > region, A mutational site, this screening enrichment can be before building sequencing library, build in sequencing library process, or carry out after building sequencing library.According to one embodiment of present invention, for sample of nucleic acid, build nucleic acid sequencing library and further comprise: utilize SLC25A13 gene mutation site Auele Specific Primer, sample of nucleic acid is carried out to pcr amplification; And for resulting amplified production, build nucleic acid sequencing library.Thus, can pass through pcr amplification, enrichment SLC25A13 gene is c.754G > A and c.1177+1G > region, A mutational site, thus can further improve the efficiency of the biological sample of screening susceptible NICCD disease.According to embodiments of the invention, the sequence of SLC25A13 gene mutation site Auele Specific Primer is not particularly limited, according to a preferred embodiment of the invention, the sequence of these SLC25A13 gene mutation site Auele Specific Primers has the nucleotide sequence as shown in SEQ ID NO:3-6:
C.754G > A mutational site primer sequence
Forward primer: ATTCGCTCCTTAACAACATGGAACTCA(SEQ ID NO:3);
Reverse primer: CAAACTGCTGGGATTTCAGGTGTGA(SEQ ID NO:4),
C.1177+1G > A mutational site primer sequence
Forward primer: CTGTTGGAGCCACTGCTGTGTATC(SEQ ID NO:5);
Reverse primer: CTTGCTAATTCATGTCAGGCACTAAGATG(SEQ ID NO:6).
Contriver is surprised to find, and by adopting these primers, can effectively realize to SLC25A13 gene c.754G > A and c.1177+1G > amplification of region, A mutational site, and the homogeneity of amplification is good.It should be noted that, the nucleotide sequence shown in these SEQ ID NO:3-6 be the present inventor after having paid arduous labor, unexpected obtain.
About for sample of nucleic acid; build method and the flow process of sequencing library; those skilled in the art can suitably select according to different sequencing technologies; details about flow process; can be referring to manufacturer's rules that for example Illumina company provides of order-checking instrument, for example, referring to the Multiplexing Sample Preparation Guide(Part#1005361 of Illumina company; Feb 2010) or Paired-End SamplePrep Guide(Part#1005063; Feb 2010), by reference, be incorporated to herein.According to embodiments of the invention, the method and apparatus from extraction from biological material sample of nucleic acid, is also not particularly limited, and can adopt commercial nucleic acid extraction kit to carry out.
It should be noted that, broad understanding should be made in the term that here used " nucleotide sequence ", it can be after the sequencing data that obtains that sample of nucleic acid is checked order is assembled, the complete nucleic acid sequence information obtaining, also can be directly to adopt by resulting sequencing data (Reads) that sample of nucleic acid is checked order as nucleotide sequence, as long as the encoding sequence that contains corresponding SLC25A13 in these nucleotide sequences.
Finally, after the nucleotide sequence of definite kernel acid sample, the sequence of the nucleotide sequence of resulting sample of nucleic acid and SEQ ID NO:1 is compared.If have and be selected from c.754G in resulting nucleotide sequence > A and c.1177+1G > at least one sudden change, indicator organism sample susceptible NICCD disease of A.Thus, by according to the method for the biological sample of the screening susceptible NICCD disease of the embodiment of the present invention, can effectively screen the biological sample of susceptible NICCD disease.According to embodiments of the invention, the method and apparatus that nucleotide sequence and SEQ ID NO:1 are compared is also not particularly limited, and can adopt the software of any conventional to operate, and according to specific examples of the present invention, can adopt SOAP software to compare.
It should be noted that, according to the purposes of " method of the biological sample of screening susceptible NICCD disease " of the embodiment of the present invention, be not particularly limited, for example can be as the screening method of non-diagnostic purpose.
System and the test kit of the biological sample of screening susceptible NICCD disease
According to a forth aspect of the invention, the present invention proposes a kind of system of method of the biological sample that can effectively implement above-mentioned screening susceptible NICCD disease.
With reference to figure 1, according to embodiments of the invention, the system 1000 of the biological sample of this screening susceptible NICCD disease comprises nucleic acid-extracting apparatus 100, nucleotide sequence determining 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.As previously mentioned, according to embodiments of the invention, the type of sample of nucleic acid is also not particularly limited, and for adopting RNA as sample of nucleic acid, nucleic acid-extracting apparatus further comprises RNA extraction unit 101 and reverse transcription unit 102, wherein, extraction unit 101 is for from extraction from biological material RNA sample, and reverse transcription unit 102 is connected with RNA extraction unit 101, for RNA sample is carried out to reverse transcription reaction, to obtain cDNA sample, resulting cDNA composition of sample sample of nucleic acid.
According to embodiments of the invention, nucleotide sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for sample of nucleic acid is analyzed, so that the nucleotide sequence of definite kernel acid sample.As previously shown, can adopt the nucleotide sequence of the method definite kernel acid sample of order-checking.Thus, according to one embodiment of present invention, described nucleotide sequence determining device 200 may further include: 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 being checked order in nucleic acid sequencing library, to obtain the sequencing result consisting of a plurality of sequencing datas.As previously mentioned, can pass through pcr amplification, enrichment SLC25A13 gene is c.754G > A and c.1177+1G > region, A mutational site, further improve the efficiency of the biological sample of screening susceptible NICCD disease.Thus, library construction unit 201 may further include pcr amplification module (not shown), in this pcr amplification module, be provided with SLC25A13 gene mutation site Auele Specific Primer, to utilize SLC25A13 gene mutation site Auele Specific Primer, described sample of nucleic acid is carried out to pcr amplification, according to a particular embodiment of the invention, SLC25A13 gene mutation site Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:3-6.According to embodiments of the invention, order-checking unit 202 can comprise and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.Thus, in conjunction with up-to-date sequencing technologies, for Single locus, can reach the higher order-checking degree of depth, detection sensitivity and accuracy improve greatly, thereby can utilize the high-throughput of these sequencing devices, the feature of degree of depth order-checking, further improve sample of nucleic acid is detected to the efficiency of analyzing.Thereby, improve follow-up accuracy and accuracy when sequencing data is analyzed.
According to embodiments of the invention, judgment means 300 is connected with nucleotide sequence determining device 200, be suitable for the nucleotide sequence of sample of nucleic acid to compare, so that the difference of the nucleotide sequence based on sample of nucleic acid and SEQ ID NO:1 judges whether susceptible NICCD disease of biological sample.Particularly, whether the nucleotide sequence based on sample of nucleic acid or its complementary sequence are compared with SEQ ID NO:1, have and be selected from c.754G > A and c.1177+1G > at least one sudden change of A, judge whether susceptible NICCD disease of biological sample.As previously mentioned, according to one embodiment of present invention, the nucleotide sequence of sample of nucleic acid or its complementary sequence are compared with SEQ ID NO:1, have and are selected from c.754G > A and c.1177+1G > at least one sudden change of A is the indication of biological sample susceptible NICCD disease.As previously mentioned, according to embodiments of the invention, the equipment that nucleotide sequence or its complementary sequence and SEQ ID NO:1 are compared is also not particularly limited, and can adopt the software of any conventional to operate, according to specific examples of the present invention, can adopt SOAP software to compare.
Thus, utilize this system, can effectively implement the method for the biological sample of aforementioned screening susceptible NICCD disease, thereby can effectively screen the biological sample of susceptible NICCD disease.
According to a fifth aspect of the invention, the present invention proposes a kind of for screening the test kit of the biological sample of susceptible NICCD disease.According to embodiments of the invention, should comprise for screening the test kit of the biological sample of susceptible NICCD disease: the reagent that is suitable for detecting SLC25A13 gene mutation body, wherein compare with SEQ ID NO:1, this SLC25A13 gene mutation body have be selected from c.754G > A and c.1177+1G > at least one sudden change of A.Utilize test kit according to an embodiment of the invention, can effectively screen the biological sample of susceptible NICCD disease.In this article, the term using " is suitable for detecting the reagent of SLC25A13 gene mutation body " and should be interpreted broadly, can be the reagent that detects SLC25A13 mutant code gene, also can be the reagent that detects SLC25A13 mutant polypeptide, for example, can adopt the antibody in identification specificity site.According to one embodiment of present invention, described reagent is nucleic acid probe, thus, can screen efficiently the biological sample of susceptible NICCD disease.
It should be noted that, in the feature and advantage of screening herein above described in the method part of biological sample of susceptible NICCD disease, be equally applicable to screen system or the test kit of the biological sample of susceptible NICCD disease, do not repeat them here.
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiment are only illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means adopting in embodiment is well known to those skilled in the art, can carry out with reference to " molecular cloning experiment guide " third edition or related products, and the reagent adopting and product are also and can business obtain.Various processes and the method do not described in detail are the ordinary methods of public office in this area, the source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all when occurring first, indicate, identical reagent used is if no special instructions, all identical with the content of indicating first thereafter.
Embodiment catches 1 target area order-checking and determines mutational site
Sample collection:
Gather the half years old young girl who suffers from tyrosinemia, galactosemia or Citrin deficiency symptoms under a cloud clinically and father and mother thereof, uncle's peripheral blood, and utilize QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany) extract respectively genomic dna as sample.Wherein, this patient's clinical manifestation is: whole skin severe xanthochromia, and color is without dark and gloomy, and other have no, the acting normally of look and spiritual aspect, sclera moderate xanthochromia, belly is the about 2.5cm of bulge ，Gan bottom right rib slightly, and matter is soft, and about 2cm under the left rib of spleen, in matter.Laboratory examination: bile acide, total bilirubin, bilirubin direct, unconjugated bilirubin raise, albumin, sphaeroprotein reduce.From clinical, this patient is suspect to be tyrosinemia, galactosemia or Citrin deficiency symptoms, but cannot make a definite diagnosis.In addition, the present embodiment has also gathered 135 normal peoples' peripheral blood and has adopted aforesaid method to extract its genomic dna sample, in contrast.
By the patient of above-mentioned collection and father and mother thereof, and normal people's genomic dna sample carry out target area catch order-checking and data analysis, specific practice is as follows:
First, contriver checks order to the target area sequence of each genomic dna sample in conjunction with Solexa high throughput sequencing technologies with NimbleGen 2.1M Human Exome Array, specific as follows:
1) chip
The chip that the present embodiment adopts is NimbleGen 2.1M Human Exome Array.
2) library construction and high-flux sequence
Each genomic dna sample is broken at random to the fragment of about 150-200bp, the process specifications providing according to manufacturers subsequently, at fragment two ends, connect respectively top connection prepare library (can be referring to:
http:// www.illumina.com/the Illumina/Solexa standard providing is built storehouse specification sheets, by reference, it is incorporated to herein in full).Linear amplification and Custom Capture Array through Ligation-mediated PCR (LM-PCR) after library is purified are hybridized enrichment, pass through again the linear amplification of LM-PCR, the order-checking that is available on the machine after library detection is qualified, to obtain primitive sequencer data.
Particularly, according to following steps, each genomic dna sample is carried out to library construction and order-checking processing:
Utilize ultrasonoscope (CovarisS2, Massachusetts, USA) respectively each genomic dna to be broken at random to the fragment of 150-200bp.Respectively get respectively 1 μ g(quantitative with Nanodrop) the Klenow fragment of T4DNA polysaccharase, T4 phosphoric acid nucleoside acid kinase and Escherichiacoli archaeal dna polymerase for DNA fragmentation of purifying processes, fills 5 ' protruding terminus and remove 3 ' protruding terminus.After adding A base, 3 ' end reconnects joint sequence.Add after joint, utilize the PCR primer that comprises 8bp sequence label (Barcode Sequence) to carry out the linear amplification of the LM-PCR of 4 circulations, to obtain the library of each genomic dna sample.Jiang Ge mixes in library afterwards and catches chip hybridizes 72h, then, utilizes 300ml elution buffer (Qiagen, Valencia, CA, USA) to carry out wash-out to the DNA fragmentation being attached on chip.Particularly, on wash-out equipment, at 95 ℃, carry out wash-out 20min, then add 200ml elution buffer and once repeat wash-out, to obtain hybridization product.The hybridization product of acquisition is carried out to the linear amplification of LM-PCR, and after library detection is qualified, with reference to the cluster of Illumina standard and the operation scheme of order-checking, utilize Illumina Hiseq2000 to check order to it, reading length is 90bp.
3) variation detection, annotation and database comparison
Utilize the primitive sequencer data of 1.7 pairs of above-mentioned acquisitions of Illumina Basecalling Software to process, filter inferior quality reads and comprise joint and pollute reads, to obtain sequencing result.Wherein, inferior quality reads comprises: reads, the mass value that surpasses 10% N base is less than 5 base and accounts for more than 50% reads of reads length, and the average quality value reads that is less than 10.After obtaining sequencing result, to nonsynonymous mutation, acceptor splicing site/donor site sudden change, insert coding region and the most possible sudden change relevant to pathology of this three class of deletion mutantion studied.Particularly, use the BWA(can be referring to: Li et al.Bioinformatics 2010.26 (5): 589-595, by reference, it is incorporated to herein in full) high quality reads is compared with reference to genome, thus, obtain the unique comparison reads comparing on genome.Then utilize the SOAPsnp (can be referring to: 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 with reference to it is incorporated to herein in full) and GATK(can be referring to: McKenna et al.Genome Res.2010.20 (9): 1297-1303, by with reference to it being incorporated in full to this paper) carry out respectively the detection of SNP and Indel.
Then, for the SNP detecting and Indel variation, contriver annotates it, i.e. definitive variation genome environment of living in, and the type of variation.Then, by the variation detecting and dbSNP database, thousand human genome databases, HapMap database, the public database such as HGMD and LSMD compares, thereby selected the variation comprising in HGMD and LSMD database and be positioned at coding region and the variation of previously not reporting in splice site district, and candidate on the known Disease-causing gene of disease is cured the disease to suddenly change and carry out Sanger checking, result, the known Disease-causing gene HPD of tyrosinemia patient, TAT and FAH, and on the known Disease-causing gene GALT of galactosemia, all do not find pathogenic mutation, but find c.754G on the known Disease-causing gene SLC25A13 of NICCD A and c.1177+1G > compound heterozygous mutations (phraseology here used " compound heterozygous mutations " of A, refer to that pair of alleles carries respectively different sudden changes).And, on patient's a allelotrope c.754G > A suddenlys change from its father, before this sudden change, be not in the news relevant with NICCD, and all predict that by SIFT and Polyphen2 software this sudden change can affect protein function; On another allelotrope of patient c.1177+1G > A suddenlys change from its mother, and on its uncle, also has this sudden change.And in 135 normal peoples' SLC25A13 gene, do not find that there is the existence of said mutation.Show thus, this patient suffers from NICCD disease, and this disease be by SLC25A13 gene c.754G > A and c.1177+1G > due to the compound heterozygous mutations of A.
Embodiment 2 Sanger method sequence verification
The genomic dna sample that utilizes patient and father and mother and uncle's (patient's father and mother and uncle are non-NICCD Disease) of embodiment 1 collection, carries out Sanger method sequence verification according to following steps to the patient's of above-mentioned acquisition SLC25A13 gene mutation site:
2.1 design of primers and PCR reaction
First, reference men and women's genoid data unit sequence storehouse hg19/build36.3, design obtains SLC25A13 gene mutation site Auele Specific Primer as follows:
C.754G > A mutational site primer sequence
Forward primer: ATTCGCTCCTTAACAACATGGAACTCA(SEQ ID NO:3);
Reverse primer: CAAACTGCTGGGATTTCAGGTGTGA(SEQ ID NO:4),
C.1177+1G > A mutational site primer sequence
Forward primer: CTGTTGGAGCCACTGCTGTGTATC(SEQ ID NO:5);
Reverse primer: CTTGCTAATTCATGTCAGGCACTAAGATG(SEQ ID NO:6).
Thus, utilize above-mentioned SLC25A13 gene mutation site Auele Specific Primer to carry out pcr amplification to genomic dna sample, in enrichment sample, SLC25A13 gene is c.754G effectively > A and c.1177+1G > region, A mutational site.
2.2PCR reaction
Then, configure respectively according to the following ratio the PCR reaction system of each genomic dna sample:
Reaction system: 25 μ L
Then, each PCR reaction system is carried out respectively to PCR reaction according to following reaction conditions:
Thus, obtain patient and father and mother and uncle's pcr amplification product.Then, by DNA Gel/PCR Purification Miniprep kit(Biomiga for each pcr amplification product obtaining, US, San Diego) cut glue and reclaim test kit and carry out respectively purifying, standby.
The order-checking of 2.3Sanger method and the result
Each obtained purified pcr amplification product is carried out respectively to DNA sequencing, to obtain sequencing result.Wherein the concrete steps of DNA sequencing are as follows: first, prepare the order-checking pre-reaction system of 10 μ L, wherein the proportioning of this 10 μ L sequencing reaction system is as follows: the purified pcr amplification product of 1 μ L, 4 μ L2.5 * BDT, 1 μ L primer (3.2pmol/ μ L), 4 μ L ultrapure waters.Then, by the sequencing reaction system preparing on PCR instrument, according to the pre-reaction of checking order of following reaction conditions: 96 ℃ of 1min; 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, 25 circulations; Last 4 ℃ of preservations, thus, obtain order-checking pre-reaction product.Next, according to following steps, order-checking pre-reaction product is carried out to purifying: a) every pipe adds 1 μ L125mM EDTA, 1 μ L3M NaAc, is added to the pipe end; B) add 25 μ L100% alcohol, obturage, shake 4 times, room temperature is placed 15min; C), in 3000x g, carry out centrifugal 30min at 4 ℃, and be inverted immediately 384 orifice plates, the centrifugal whizzer power supply of closing during to 185x g; D) add 35 μ L70% alcohol, 3000x g, carries out centrifugal 15min at 4 ℃, and is inverted immediately 384 orifice plates, the centrifugal whizzer power supply of closing during to 185x g; E) repeat 70% alcohol washing 1 time; F) remaining alcohol is volatilized under room temperature and do, then add 10 μ LHi-Di methane amides, at 95 ℃, carry out after sex change 4min rapid ice-cold 4min.Then, purified order-checking pre-reaction product is gone up rapidly to ABI3730xl sequenator and check order, to obtain sequencing result.
Then,, based on sequencing result, utilize 5.2 couples of patients of Sequencing Analysis of ABI and father and mother thereof and uncle to carry out the comparison of SLC25A13 gene coded sequence and analysis.Wherein, to patient and father and mother and uncle's SLC25A13 gene mutation site c.1177+1G Fig. 2 and Fig. 3 have shown respectively > A and c.754G > the Sanger sequence verification peak figure (as shown in the figure, heterozygosis peak, arrow place overlaps or partially overlapped) of A.In conjunction with Fig. 2 and Fig. 3, contriver finds to have c.1177+1G on patient's a allelotrope > heterozygous mutant of A, on another allelotrope, have c.754G > A(p.Thr539Ile) heterozygous mutant; C.1177+1G patient's mother and uncle have > heterozygous mutant of A; C.754G patient's father has > A(p.Thr539Ile) heterozygous mutant.Thus, contriver checking, confirmed c.1177+1G > A with c.754G > A is the new mutant site relevant to NICCD disease incidence.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the feature of this embodiment or example description.In this manual, the schematic statement of above-mentioned term is not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or feature can be with suitable mode combinations in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. a nucleic acid for separated coding SLC25A13 mutant, is characterized in that, compares with SEQ ID NO:1, and described nucleic acid has and is selected from least one following sudden change: c.754G > A and c.1177+1G > A,
Preferably, c.754G described nucleic acid have simultaneously > A and c.1177+1G > A sudden change,
Optionally, described nucleic acid is DNA.
2. an isolated polypeptide, is characterized in that, compares with SEQ ID NO:2, and described isolated polypeptide has and is selected from least one following sudden change: p.Glu252Lys and p.Gly393Glyfs*2,
Preferably, described polypeptide has p.Glu252Lys and p.Gly393Glyfs*2 sudden change simultaneously,
Optionally, described polypeptide is by nucleic acid encoding claimed in claim 1.
3. a method of screening the biological sample of susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases, is characterized in that, comprises the following steps:
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 sequence are compared with SEQ ID NO:1, have and are selected from c.754G > A and c.1177+1G > at least one sudden change of A is the indication of described biological sample susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases,
Optionally, described biological sample is to be selected from least one of blood of human body, skin, hair and muscle,
Optionally, described sample of nucleic acid is complete genome DNA.
4. method according to claim 3, is characterized in that, from described extraction from biological material sample of nucleic acid, further comprises:
From described extraction from biological material RNA sample, preferred described RNA sample is mRNA; And
Based on described RNA sample, by reverse transcription reaction, obtain cDNA sample, sample of nucleic acid described in described cDNA composition of sample.
5. method according to claim 3, is characterized in that, determines that the nucleotide sequence of described sample of nucleic acid further comprises:
For described sample of nucleic acid, build nucleic acid sequencing library; And
Checked order in described nucleic acid sequencing library, to obtain the sequencing result formed by a plurality of sequencing datas, optionally, adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in described nucleic acid sequencing library,
Optionally, for described sample of nucleic acid, build nucleic acid sequencing library and further comprise:
Utilize SLC25A13 gene mutation site Auele Specific Primer, described sample of nucleic acid is carried out to pcr amplification; And
For resulting amplified production, build described nucleic acid sequencing library,
Optionally, described SLC25A13 gene mutation site Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:3-6.
6. a system of screening the biological sample of susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases, is characterized in that, comprising:
Nucleic acid-extracting apparatus, described nucleic acid-extracting apparatus is used for from described extraction from biological material sample of nucleic acid;
Nucleotide sequence determining device, described nucleotide sequence determining device is connected with described nucleic acid-extracting apparatus, for described sample of nucleic acid is analyzed, to determine the nucleotide sequence of described sample of nucleic acid;
Judgment means, described judgment means is connected with described nucleotide sequence determining device, so that the nucleotide sequence based on described sample of nucleic acid or its complementary sequence are compared with SEQ ID NO:1, whether have and be selected from c.754G A and c.1177+1G > at least one sudden change of A, judge whether susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases of described biological sample.
7. system according to claim 6, is characterized in that, described nucleic acid-extracting apparatus further comprises:
RNA extraction unit, described RNA extraction unit is used 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 described RNA sample is carried out to reverse transcription reaction, to obtain cDNA sample, sample of nucleic acid described in described cDNA composition of sample.
8. system according to claim 6, is characterized in that, described nucleotide sequence determining device further comprises:
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 being checked order in described nucleic acid sequencing library, to obtain the sequencing result being formed by a plurality of sequencing datas,
Optionally, described library construction unit further comprises:
Pcr amplification module, is provided with SLC25A13 gene mutation site Auele Specific Primer in described pcr amplification module, to utilize SLC25A13 gene mutation site Auele Specific Primer, described sample of nucleic acid is carried out to pcr amplification,
Optionally, described SLC25A13 gene mutation site Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:3-6,
Optionally, described order-checking unit comprises and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.
9. for screening a test kit for the biological sample of susceptible newborn infant melon amino acid mass formed by blood stasis 2 type diseases, it is characterized in that, contain:
Be suitable for detecting the reagent of SLC25A13 gene mutation body, wherein compare with SEQ ID NO:1, described SLC25A13 gene mutation body have be selected from c.754G > A and c.1177+1G > at least one sudden change of A.
10. test kit according to claim 9, is characterized in that, described reagent is nucleic acid probe.
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| CN101993952A (en) * | 2009-08-21 | 2011-03-30 | 复旦大学附属儿科医院 | Mutation screening probe of SLC25A13 gene and application thereof |
| CN102154509A (en) * | 2010-12-28 | 2011-08-17 | 深圳华大基因科技有限公司 | Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation |
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| CN101993952A (en) * | 2009-08-21 | 2011-03-30 | 复旦大学附属儿科医院 | Mutation screening probe of SLC25A13 gene and application thereof |
| CN102154509A (en) * | 2010-12-28 | 2011-08-17 | 深圳华大基因科技有限公司 | Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation |
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