CN111961713A - Probe composition and kit for screening carriers of pathogenic genes of genetic diseases and preparation method of probe composition and kit - Google Patents
Probe composition and kit for screening carriers of pathogenic genes of genetic diseases and preparation method of probe composition and kit Download PDFInfo
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Abstract
The invention provides a probe composition and a kit for screening carriers of pathogenic genes of genetic diseases and a preparation method thereof. The probe composition specifically captures pathogenic genes of genetic diseases, wherein the genetic diseases comprise alpha-thalassemia, beta-thalassemia, spinal muscular atrophy, hepatolenticular degeneration and methylmalonic acidemia cblC type. The invention provides a probe combination which is accurate, reliable, simple and economic and can simultaneously screen carriers of pathogenic genes of various genetic diseases, and is convenient for popularization and clinical application of screening of carriers of common genetic diseases.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a probe composition and a kit for screening carriers of pathogenic genes of genetic diseases and a preparation method thereof.
Background
Monogenic genetic diseases refer to genetic diseases controlled by a pair of alleles, such as Spinal Muscular Atrophy (SMA), thalassemia, hepatolenticular degeneration, methylmalonic acidemia, and the like. There are many kinds of monogenic hereditary Diseases, and 9000 kinds of monogenic hereditary Diseases (http:// www.omim.org/statistics/entry) are found at present, the comprehensive morbidity is as high as 1/100(https:// www.who.int/genetics/public/genetic Diseases/en/index2. html), most monogenic hereditary Diseases are lethal, teratogenic or disabling, only 5% of the effective therapeutic DRUGs (ORPHAN DRUG REPORT 2014) are available, and the treatment cost is high Often, the condition is not discovered until after birth. If the carrier of the monogenic genetic disease is screened in the pregnancy preparation period or the early pregnancy period, the risk of the child bearing child is found in time, and the occurrence of serious genetic disease can be effectively prevented by combining the measures of genetic consultation, prenatal detection or diagnosis, assisted reproduction and the like, thereby having important significance for reducing birth defects and improving the quality of the birth population.
At present, no kit which is suitable for phenotype normal population and can simultaneously carry out carrier screening on multiple single-gene disease genes exists clinically, and few kits based on second-generation sequencing and probe capture have poor probe capture uniformity. In addition, the capture area of the probe in the existing related kit is not properly selected, and the capture area is too much, so that the quantity of the probe is large, and the detection cost is high. And a few of kits for screening monogenic disease carriers have a single disease detection range, for example, the kit for screening SMA pathogenic gene carriers adopts a fluorescent quantitative PCR method, the products have limited disease screening, and the detection method has the defects of low sensitivity and low specificity. The most expected application of the common monogene detection kits in the market is clinical diagnosis of patients, the kits generally adopt the traditional detection methods, for example, the thalassemia detection methods comprise Gap-PCR, PCR-RDB, realtime PCR, sanger sequencing and the like, the SMA mutation detection methods comprise QPCR, Multiple Ligation Probe Amplification (MLPA) and the like, the defects of low flux, low sensitivity, limited detection range and the like exist, and the kit is not suitable for screening of carriers of large groups.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a probe composition for screening carriers of pathogenic genes of genetic diseases, a gene chip and a preparation method thereof.
According to a first aspect, there is provided in one embodiment a probe composition that specifically captures the causative genes of genetic diseases including, but not limited to, a-thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, methylmalonic acidemia cblC type.
According to a second aspect, there is provided in one embodiment a method of preparing a probe composition for screening carriers of a genetic disease causing gene, comprising:
a target capture region selection step of selecting respective target capture regions according to the target genes;
and designing probes according to the target capture region, wherein the lengths of the capture probes in the non-homologous regions of the pathogenic genes in the target capture region are the same, and the length of the capture probe in the partial homologous region of the pathogenic genes in the target capture region is 110-. The purpose of the partial homology region capture probes being less than 120nt in length is to improve the specificity of the capture probes in these regions.
According to a third aspect, an embodiment provides a gene chip on which the probe composition according to the first aspect or the probe composition prepared by the preparation method according to the second aspect is immobilized.
According to a fourth aspect, there is provided in one embodiment a kit comprising a probe composition according to the first aspect, or a probe composition prepared by the preparation method according to the second aspect, or a gene chip according to the third aspect.
According to a fifth aspect, an embodiment provides a detection device comprising the probe composition according to the first aspect, or the probe composition prepared by the preparation method according to the second aspect, or the gene chip according to the third aspect, or the kit according to the fourth aspect.
According to a sixth aspect, there is provided in one embodiment a method of sequencing comprising: capturing the target region using the probe composition according to the first aspect, or the probe composition prepared by the preparation method according to the second aspect, or the gene chip according to the third aspect, or the kit according to the fourth aspect, or the detection device according to the fifth aspect.
The invention has the beneficial effects that:
1. the length of each synthesized probe is fixed, and furthermore, the method is accurate and reliable through independent synthesis and independent quality control;
2. when the kit contains probes capable of capturing various disease pathogenic genes, carriers of various genetic disease pathogenic genes can be screened simultaneously, and the kit is simple and economical and is convenient for popularization and clinical application of screening of common genetic disease carriers.
Drawings
FIG. 1 is a flow chart of variation detection and analysis according to an embodiment of the present invention.
FIG. 2 is a statistical chart showing the uniformity of coverage of capture regions for two protocols of synthetic probes in example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
In some embodiments, the present invention provides a method and kit capable of screening 5 common genetic disease genes simultaneously for phenotypically normal populations. By adopting the method and the kit, through fragmenting, fragment screening, end repairing, fragment enriching, probe capturing and sequencing the DNA sample and combining corresponding variation analysis software, the method and the kit can simultaneously detect the related gene variation of alpha-thalassemia, beta-thalassemia, SMA, hepatolenticular degeneration and cblC type methylmalonemia, realize the carrier screening of various common genetic diseases based on one method, effectively simplify the detection process, improve the detection flux and the accuracy of detection and reduce the detection cost compared with the carrier screening products of single diseases based on the traditional method.
In a first aspect, the present invention provides in some embodiments a probe composition that specifically captures the causative genes of genetic diseases including, but not limited to, α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, methylmalonatemia cblC type.
In some embodiments, the length of each probe in the capture probe composition of the non-homologous region of the disease-causing gene is the same.
In some embodiments, the partial homology region capture probe for the causative genes of α -thalassemia, β -thalassemia, spinal muscular atrophy is 110-120nt in length. The purpose of the partial homologous region capture probe length being less than 120nt is to increase the specificity of the capture probe in these regions
In some embodiments, each probe in the capture probe composition of the non-homologous region of the disease-causing gene is 120nt in length.
In some embodiments, the fault tolerance of each probe in the probe composition is < 7 nt.
In some embodiments, the coverage density of each probe in the probe composition is 1 x.
In some embodiments, the target capture area of the probe composition includes, but is not limited to, at least one of:
(a) the pathogenic genes related to alpha-thalassemia HBA1 and HBA 2;
(b) beta-thalassemia-associated virulence gene HBB (beta globin gene);
(c) spinal Muscular Atrophy (SMA) -associated virulence gene SMN 1;
(d) pathogenic gene ATP7B related to hepatolenticular degeneration;
(e) the pathogenic gene MMACHC related to the methyl malonic acidemia cblC type.
In some embodiments, for region (a), the full length fragments of the pathogenic genes HBA1 and HBA2 are targeted and extended 50bp each upstream and downstream as the final target capture region.
In some embodiments, in said region (a), for-SEAAbsence, -alpha3.7Absence, -alpha4.2Deletion, selecting 1 SNP site every 1kb in the deletion range as a target capture region.
In some embodiments, for said region (b), the HBB full-length fragment of the disease-causing gene is targeted and extended 50bp each upstream and downstream as the final target capture region.
In some embodiments, in the region (b), 1 SNP site is selected as the target capture region every 1kb within the deletion range for mainland china deletion, taiwan deletion, SEA-HPFH deletion.
In some embodiments, for region (c), the whole genome region of the disease-causing gene SMN1 is targeted for capture.
In some embodiments, for the region (d), the exon region of the causative gene ATP7B is used as the final target capture region.
In some embodiments, for said region (e), the exon region of the disease-causing gene MMACHC is taken as the final target capture region.
In some embodiments, each probe in the probe composition is a single-stranded nucleotide sequence, which may be a single-stranded DNA or a single-stranded RNA, preferably a single-stranded DNA.
In some embodiments, the probe composition comprises 1270 nucleotide sequences that specifically capture at least one of the alpha-thalassemia-associated virulence genes HBA1 and HBA2, beta-thalassemia-associated virulence gene HBB, Spinal Muscular Atrophy (SMA) -associated virulence gene SMN1, hepatolenticular degeneration-associated virulence gene ATP7B, methylmalonemia cblC-type-associated virulence gene MMACHC, respectively.
In some embodiments, the probe composition has at least 80% sequence identity to at least one of the 1270 nucleotide sequence or its complement.
In a preferred embodiment, said probe composition comprises at least one of said 1270 nucleotide sequences, or the complement thereof.
In a more preferred embodiment, said probe composition comprises all of said 1270 nucleotide sequences, or the complement thereof.
In a second aspect, the present invention provides, in some embodiments, a method of preparing a probe composition for screening carriers of a genetic disease causing gene, comprising:
a target capture region selection step of selecting respective target capture regions according to the target genes;
and designing probes according to the target capture region, wherein the lengths of the capture probes in the non-homologous regions of the pathogenic genes in the target capture region are the same, and the length of the capture probe in the partial homologous region of the pathogenic genes in the target capture region is 110-. The purpose of the partial homology region capture probes being less than 120nt in length is to improve the specificity of the capture probes in these regions.
In some embodiments, after the probes are designed, each of the probes is synthesized separately.
In some embodiments, each of the probes is individually synthesized and then individually quality controlled.
The length of each probe is fixed, the probes are synthesized independently and are controlled independently, the accuracy of probe sequence synthesis is effectively improved, and the quality condition of each probe is unknown in the mixed probes in the prior art.
The quality control is to ensure the quality of the synthesized probe, and if the quality control is not performed, the quality of the synthesized probe is unknown, which may result in poor or no capture of the target region.
In the prior art, the length of the probe is short (such as less than 100nt), the probe is not independently synthesized and independently controlled, and the quality of each probe is unknown; in some embodiments of the invention, each probe is fixed in length, synthesized separately, and controlled separately, which effectively improves the accuracy of probe sequence synthesis.
In some embodiments, all probes are synthesized at equal concentrations.
In some embodiments, all probes are mixed in equal amounts (pooling).
In some embodiments, the fault tolerance of each of the probes is < 7 nt.
Herein, the tolerance ratio refers to a ratio that allows an erroneous base to be synthesized when a probe is synthesized.
In some embodiments, the coverage density of each of the probes is 1 x.
Herein, a coverage density of 1x means that the probes are connected end to end and cover the target area at least once.
In some embodiments, the genetic disease includes, but is not limited to, at least one of α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, and methylmalonic acidemia cblC type, and specifically may include 1, 2, 3, 4, 5 of the above genetic diseases.
In a preferred embodiment, the genetic disease comprises all diseases in α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, methylmalonic acidemia cblC type.
In a preferred embodiment, the capture probe for the partial homology region of the pathogenic genes of α -thalassemia, β -thalassemia and spinal muscular atrophy has a length of 110-120nt, and specifically may include, but is not limited to, 110nt, 111nt, 112nt, 113nt, 114nt, 115nt, 116nt, 117nt, 118nt, 119nt, 120 nt.
In some embodiments, the target capture area comprises at least one of:
(a) the pathogenic genes related to alpha-thalassemia HBA1 and HBA 2;
(b) beta-thalassemia-associated virulence gene HBB (beta globin gene);
(c) spinal Muscular Atrophy (SMA) -associated virulence gene SMN 1;
(d) pathogenic gene ATP7B related to hepatolenticular degeneration;
(e) the pathogenic gene MMACHC related to the methyl malonic acidemia cblC type.
In some embodiments, the target capture area comprises at least one of the regions (a) - (e), and specifically may comprise 1, 2, 3, 4 or 5 of the regions (a) - (e), and in some preferred embodiments, the target capture area comprises all of the regions (a) - (e).
In some embodiments, for region (a), the full length fragments of the pathogenic genes HBA1 and HBA2 are targeted and extended 50bp each upstream and downstream as the final target capture region.
In some embodiments, in said region (a), for-SEAAbsence, -alpha3.7Absence, -alpha4.2Deletion, selecting 1 SNP site every 1kb in the deletion range as a target capture region.
In some embodiments, for said region (b), the HBB full-length fragment of the disease-causing gene is targeted and extended 50bp each upstream and downstream as the final target capture region.
In some embodiments, in the region (b), 1 SNP site is selected as the target capture region every 1kb within the deletion range for mainland china deletion, taiwan deletion, SEA-HPFH deletion.
SEA-HPFH refers to the Southeast Asian heredity persistent fetal hemoglobin syndrome (SEA-HPFH).
In some embodiments, for region (c), the whole genome region of the disease-causing gene SMN1 is targeted for capture.
In some embodiments, for the region (d), the exon region of the causative gene ATP7B is used as the final target capture region.
In some embodiments, for said region (e), the exon region of the disease-causing gene MMACHC is taken as the final target capture region.
In a preferred embodiment, the target capture region comprises all of the disease causing genes of regions (a) - (e). The kit enables the detection method to be accurate, reliable, simple and economical, can simultaneously carry out carrier screening on a plurality of genetic disease pathogenic genes, and is convenient for popularization and clinical application of common genetic disease carrier screening.
In some embodiments, each probe in the probe composition is a single-stranded nucleotide sequence, which may be a single-stranded DNA or a single-stranded RNA, preferably a single-stranded DNA.
In some embodiments, the probe composition comprises at least one of 1270 nucleotide sequences that specifically capture alpha-thalassemia-associated virulence genes HBA1 and HBA2, beta-thalassemia-associated virulence gene HBB, Spinal Muscular Atrophy (SMA) -associated virulence gene SMN1, hepatolenticular degeneration-associated virulence gene ATP7B, methylmalonemia cblC-type-associated virulence gene MMACHC, or at least one of the complement of the 1270 nucleotide sequences, respectively.
For the target capture area, under the condition of being capable of completing capture, the fewer the target capture areas are, the fewer the number of probes is, and the lower the synthesis cost is, on the premise of being capable of achieving the purpose of disease detection, the invention screens the fewest target capture areas, selects the areas (a) - (e) as the target capture areas, and only 1270 probes are needed, so that the number of needed probes is effectively reduced, and the synthesis cost of the probes is further reduced.
In some embodiments, the probe composition has at least 80% sequence identity to at least one of the 1270 nucleotide sequence or its complement. In other embodiments, the probe has 80% to 100% sequence identity to at least one of the 1270 nucleotide sequence or its complement. In other embodiments, the probe composition has at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity to at least one of the 1270 nucleotide sequence or the complement thereof.
In a preferred embodiment, said probe composition comprises at least one of said 1270 nucleotide sequence, or the complement thereof.
In a more preferred embodiment, said probe composition comprises all of said 1270 nucleotide sequence, or the complement thereof.
In a third aspect, the present invention provides, in some embodiments, a gene chip on which the probe composition according to the first aspect or the probe composition prepared by the preparation method according to the second aspect is immobilized.
In a fourth aspect, the present invention provides, in some embodiments, a kit comprising a probe composition according to the first aspect, or a probe composition prepared by the preparation method according to the second aspect, or a gene chip according to the third aspect.
In a fifth aspect, the present invention provides, in some embodiments, a test device comprising a probe composition according to the first aspect, or a probe composition prepared according to the preparation method of the second aspect, or a gene chip according to the third aspect, or a kit according to the fourth aspect.
In a sixth aspect, the present invention provides in some embodiments the use of a probe composition according to the first aspect for the preparation of a gene chip, kit or assay device for the screening of carriers of a genetic disease causing gene.
In a seventh aspect, the present invention provides, in some embodiments, a sequencing method comprising: capturing the target region using the probe composition according to the first aspect, or the probe composition prepared by the preparation method according to the second aspect, or the gene chip according to the third aspect, or the kit according to the fourth aspect, or the detection device according to the fifth aspect.
In some embodiments, the above sequencing methods can be used for scientific research, such as for non-diagnostic therapeutic purposes such as screening for new drug candidates for related genetic diseases. Moreover, the sequencing method is not a diagnostic method for diseases because it is an ex vivo sample, and an intermediate reference result is obtained, but not a final result, and usually requires a combination of clinical symptoms, past medical history, family genetic history and other data of a subject to prepare for judging the disease condition of the subject.
In some embodiments, the method further comprises a step of genomic DNA fragmentation and fragment double selection, a step of phosphorylation reaction and adaptor ligation, a step of fragment enrichment, wherein after the probe composition according to the first aspect, or the probe composition prepared by the preparation method according to the second aspect, or the gene chip according to the third aspect, or the kit according to the fourth aspect, or the detection device according to the fifth aspect, the target region of the enriched fragment is captured, a sequencing result is obtained by high-throughput sequencing and mutation automated analysis.
In some embodiments, the detection method of the present invention is used for carrier screening, and determining whether the infant born by a couple is at risk of disease according to the detection result, and is not a diagnostic and therapeutic method of disease. In some embodiments, the probe, gene chip, kit or detection device and the detection method thereof can be used for screening of new drug candidates and other scientific research works for non-diagnostic treatment purposes.
In some embodiments, the step of fragment double-selecting of genomic DNA comprises:
a primary selection step, wherein magnetic beads are adopted to perform primary selection on the broken DNA fragments, and large fragments are removed to obtain primary selection products;
and a second selection step, namely, performing secondary screening on the primary selection product by adopting the magnetic beads again.
In some embodiments, in the initial selection, the desired small fragments are selected by controlling the ratio of DNA volume to the volume of added beads, and large fragments are removed.
In some embodiments, the ratio of the volume of DNA to the volume of added beads at initial selection is 5: 3.
in some embodiments, in the second option, fragments of a desired length range are also selected by controlling the ratio of the DNA volume to the volume of the added beads, and other fragments are removed.
In some embodiments, the ratio of DNA volume to added bead volume is 20: 3.
in some embodiments, the invention provides a method and a kit capable of screening 5 common genetic diseases aiming at normal people, wherein the 5 common genetic diseases are alpha-thalassemia, beta-thalassemia, SMA, hepatolenticular degeneration and methyl malonic acidemia cblC types, and the detection method comprises the steps of fragmentation and fragment double selection of a DNA sample, phosphorylation reaction and adaptor connection, fragment enrichment, probe design and capture, sequencing and mutation analysis. The mutation detection process is shown in FIG. 1.
In some embodiments, the invention provides kits and methods for simultaneously screening 5 genetic diseases including α -thalassemia, β -thalassemia, SMA, hepatolenticular degeneration and methylmalonemia cblC type.
In one embodiment, the method for simultaneously screening 5 common genetic diseases for a normal population provided by the invention comprises the following steps:
1. fragmentation and fragment double selection of genomic DNA
The genome DNA is broken by DNA fragmenting enzyme, and the length range of the fragmented DNA is controlled by controlling the addition amount and the reaction time of the DNA fragmenting enzyme; and (2) screening the broken DNA fragments by using magnetic beads, firstly, primarily selecting, screening large fragments by controlling the proportion of the volume of the DNA to the volume of the added magnetic beads, secondly, secondarily selecting, adding the magnetic beads into primarily selected products, and recovering the DNA fragments in a certain size range by controlling the proportion of the volume of the DNA to the volume of the added magnetic beads, thereby realizing the screening control of fragmented DNA in a certain size.
2. Phosphorylation reaction and linker attachment
Since the fragmented product has no phosphate group at the 5 'end, before ligation of the tag linker, phosphokinase (e.g., T4 polynucleotide phosphokinase) is used to modify the 5' end of the fragmented product by phosphorylation, and phosphate group is introduced to facilitate linker ligation.
The DNA after phosphorylation is connected with a label joint under the action of ligase, and the label joint contains a structural sequence required by sequencing cyclization and a sequencing primer binding region required by sequencing reaction, so that the product can be guided to carry out cyclization, DNB (DNA nanosphere) preparation and sequencing reaction.
3. Fragment enrichment
And performing PCR amplification on the adaptor-connected fragmented DNA, enriching the adaptor-connected fragmented DNA with a specific length range, and obtaining a single-sample library.
4. Probe design and probe capture
4.1 Probe design
1) Selection of target region:
a. alpha-thalassemia related pathogenic genes HBA1 and HBA2, aiming at full-length gene fragments and extending the gene fragments by 50bp upwards and downwards as final target capture regions, for common-SEAAbsence, -alpha3.7Absence, -alpha4.2Deletion, selecting 1 SNP site every 1kb in the deletion range as a target capture region.
b. Aiming at a gene full-length fragment, a pathogenic gene HBB (beta globin gene) related to the beta-thalassemia extends 50bp upwards and downwards to be used as a final target capture region, and for common mainland China type deletion, Taiwan type deletion and SEA-HPFH deletion, 1 SNP locus is selected every 1kb in a deletion range to be used as a target capture region.
The whole genome region of the SMA-related pathogenic gene SMN1 is used as a target capture region.
d. The exon region of ATP7B, a pathogenic gene related to hepatolenticular degeneration, serves as a final target capture region.
e. The MMACHC exon region of the pathogenic gene related to the clobc type of the methylmalonic acidemia serves as a final target capture region.
2) Design and Synthesis of probes
a. The length of the non-homologous region capture probe is 120nt, the length of the alpha-thalassemia, beta-thalassemia and SMA gene partial homologous region capture probe is 110-120nt, and the target region is specifically identified;
b. the fault tolerance rate of each probe is less than 7 nt;
c. the probe coverage density was 1 x;
d. the Hits number between the probe and the reference genome is less than 30.
In one embodiment, the Target region in step 1) is designed by using Target Capture Probe Design & Ordering Tool Design software (https:// sg. idtdna. com/site/order/ngs), and the Design parameters of the software are set as Target specifications: Human (Feb.2009 GRCh 37/hg19), Probe length:120 and Probe timing sensitivity: 1x, and the probes with the Hits number of the Probe sequence and the reference genome being more than 30 are removed.
After the above condition design, the number of the probes in the a-e region in the capturing step 1) is 1270, when the probes are synthesized, each probe is synthesized by adopting a chemical synthesis method, then each probe is subjected to quality control by adopting a mass spectrometry method, the probes with the synthesized probe length smaller than the corresponding designed length are synthesized again, the 1270 probes are synthesized in an equal concentration, then equal mixing (discharging) is carried out, and the synthesis and the quality control of the probes are entrusted to Integrated DNA Technologies (IDT).
The 1270 probe sequences are shown in Table 1 and are sequentially marked as SEQ ID No. 1-SEQ ID No.1270, and the sequence directions in Table 1 are all 5 'to 3':
TABLE 1
In Table 1, the probes less than 120nt (specifically, 111nt to 119nt) are capture probes for α -thalassemia, β -thalassemia, and regions homologous to Spinal Muscular Atrophy (SMA) causative gene, and the length of the probes less than 120nt is intended to improve the specificity of the capture probes in these regions.
3) Probe capture
Pre-hybridization library treatment: mixing the single sample libraries in the step 3 (n is less than or equal to 40), adding the hybrid COTDNA and the closed linker sequence, and concentrating the mixture. The concentrated mixture is pre-deformed to form single-stranded DNA, and the COTDNA and the closed joint sequence respectively close the repetitive sequence and the joint sequence in the hybridization process, so that the occurrence of nonspecific hybridization and secondary capture is reduced.
Probe hybridization and elution: and hybridizing the probe with the treated mixed library, and eluting and purifying the hybridized library by using a hybridization eluent and magnetic beads to obtain the hybridized library with the target region.
Enrichment and purification by PCR: enrichment of the hybridization library based on PCR, and magnetic bead purification.
5. High throughput sequencing and automated analysis of variants
Sequencing the hybridization library by using a high-throughput sequencer, wherein the average effective depth of the target area is not less than 100 x.
And (4) performing variation analysis, judgment and output on the sequencing data by using variation automatic analysis software.
The technical scheme of the invention at least has the following beneficial effects:
the kit realizes the simultaneous carrier screening of 5 common genetic diseases for the first time, fills the gap of the lack of related products for simultaneously carrying out carrier screening on multiple genetic diseases clinically, simplifies the detection process, improves the detection flux and the detection accuracy and reduces the detection cost compared with the carrier screening products of single diseases based on the traditional method. By combining with a corresponding information analysis method, the known pathogenic variation can be automatically judged and output, the manual interpretation cost of the variation is saved, and the method is suitable for large-scale screening of people. The invention is not limited to simultaneously detecting the 5 common genetic diseases, and probes for other genetic diseases can be designed according to the design principle of the invention, so that carrier screening for more genetic diseases can be realized simultaneously.
Example 1
SMA-related gene detection
The detection of deletion mutation of exon 7 of SMN1 gene related to SMA is carried out on 418 samples with known clinical results by adopting the kit and a commercial kit (Shanghai Pentahromite medicine research GmbH, SMN1) exon deletion detection kit (fluorescent quantitative PCR method)), and the detection is carried out completely according to the instruction of the commercial kit (qPCR method) based on the detection flow as described in the following (I).
Variant detection process based on kit
1. Disruption of genomic DNA
1.1 prepare the amount of the reaction mixture needed to be interrupted for the assay in the appropriate centrifuge tube according to the proportions in Table 2.
Table 2 disruption of the reaction mixture
1.2 Add 5. mu.L of the cleavage reaction mixture to the PCR tube (containing genomic DNA) of step 1.1, mix well, centrifuge, incubate at 37 ℃ for 30min on the PCR instrument, cool to 4 ℃ and keep.
1.3 after the interruption, taking out the PCR tube, placing the PCR tube on an ice box, immediately adding 5 mu L of stop solution, blowing, uniformly mixing, and reacting at room temperature for 1min to obtain an interruption product.
2. Magnetic bead double selection
2.1 Primary selection
Transferring the product obtained in the step 1.3 into a 1.5mL centrifuge tube, adding 25. mu.L of LTE elution buffer and 30. mu.L of purified magnetic beads (V) which are equilibrated to room temperatureMagnetic bead： VDNA＝0.6，VMagnetic bead、VDNARespectively referring to the volume of the magnetic beads and the DNA), placing on a vortex oscillator, fully mixing, standing at room temperature for 5min, and adsorbing by a magnetic frame toClear and carefully pipette the supernatant into a new 1.5mL centrifuge tube.
The magnetic beads used herein were purchased from Nanjing Novozam Biotechnology Inc., VAHTSDNACANDEANBEADS, Cat.N 411-03.
2.2 two options
2.2.1 adding 12 μ L of purified magnetic beads into the supernatant obtained in step 1.1, placing on a vortex oscillator, mixing well, standing at room temperature for 5min, and adsorbing by a magnetic frame until the supernatant is clear.
2.2.2 carefully aspirate the supernatant, add 200. mu.L of 75% ethanol and let stand for 1min until the liquid is clear. This step was repeated once.
2.2.3 carefully remove the supernatant, keep the centrifuge tube on the magnetic frame, open the tube cover, and dry it at room temperature until the surface of the beads is not reflective.
2.2.4 taking the centrifuge tube off the magnetic frame, adding 42 mul of elution buffer solution, placing on a vortex oscillator for fully mixing, standing for 5-10min at room temperature after instantaneous centrifugation, and adsorbing the magnetic frame until the magnetic frame is clarified and absorbing 40 mul into a new PCR tube.
3. End repair plus A
3.1 prepare the end-repair reaction mixture on ice according to the proportions of Table 3.
TABLE 3 end-repair reaction mixture
| Name of reagent | One reaction standard amount |
| End repair enzyme | 1.55μL |
| End repair buffer | 8.45μL |
| Total volume | 10μL |
In Table 3, the end repair enzymes and their buffers were purchased from Shenzhen Huazhi Daizhiji technology Co., Ltd, T4PNK (cat No. 090-.
3.2 Add 10. mu.L of the end-repair reaction mixture (ice-on) to the 2.2.4PCR tube, mix well, centrifuge instantaneously, incubate in the PCR instrument at 37 ℃ for 15min, 65 ℃ for 15min (hot lid: 105 ℃), cool to 4 ℃ and hold. After the reaction, the PCR tube was taken out and centrifuged instantaneously.
4. Joint connection
4.1 prepare ligation mixtures in the amounts required for detection on ice according to the proportions in Table 4.
TABLE 4 ligation reaction mixtures
| Name of reagent | One reaction standard amount |
| Ligase | 1.6μL |
| Ligation buffer | 23.4μL |
| Total volume | 25μL |
In Table 4, ligase and ligation buffer were purchased from Shenzhen Huazhi Zhi Tech Co., Ltd, T4 DNAIgase, cat # 090-.
And 4.2, taking out the tag joints (namely the tag joints in the library building reagent of the MGI-SEQ2000 sequencer) according to the library building quantity, oscillating, uniformly mixing, performing instantaneous centrifugation, and sequentially adding 5 mu L of tag joints into the end repair reaction products according to the sequence, wherein each sample needs to correspond to a unique tag joint.
4.3 Add 25. mu.L ligation buffer to the product of step 4.2, mix well, centrifuge instantaneously, incubate for 30min at 23 ℃ on the PCR instrument (hot lid: closed), cool to 4 ℃ and hold. And (5) taking out the PCR tube after the reaction is finished, and performing instant centrifugation.
5. Purification after linker attachment
5.1 transferring the product obtained in the step 4.3 to a 1.5mL centrifuge tube, adding 20 μ L of elution buffer solution and 50 μ L of purified magnetic beads which are balanced to room temperature, placing on a vortex oscillator, fully mixing, performing instantaneous centrifugation, standing at room temperature for 5-10min, and adsorbing by a magnetic frame until the mixture is clear.
5.2 carefully remove the supernatant by aspiration, add 200. mu.L of 75% ethanol, and let stand for 1min until the liquid is clear. This step was repeated once.
5.3 carefully absorbing and discarding the supernatant, keeping the centrifuge tube on the magnetic frame, opening the tube cover, and drying at room temperature until the surface of the magnetic beads is not reflective.
5.4 remove the centrifuge tube from the magnetic frame, add 40 μ L of elution buffer, place on vortex shaker, mix well, centrifuge instantaneously, stand for 5-10min, the magnetic frame adsorbs until clear, carefully absorb 38 μ L of supernatant into PCR tube.
Pre-PCR amplification
6.1 prepare the amount of Pre-PCR reaction mix required for detection on ice according to the proportions of Table 5.
TABLE 5Pre-PCR reaction mixture
| Name of reagent | One reaction standard amount |
| PCR reaction solution | 50μL |
| Primer and method for producing the same | 12μL |
| Total volume | 62μL |
In Table 5, the PCR reaction solution was purchased from Shenzhen Huazhiyu science and technology Limited, AlphaHigh-FidelityReadyMix, cat No. 01K00901 MM.
The primers in table 5 are as follows:
an upstream primer: 5'-GAACGACATGGCTACGA-3' (SEQ ID No. 1271);
a downstream primer: 5'-TGTGAGCCAAGGAGTTG-3' (SEQ ID No. 1272).
6.2 Add 62. mu. LPre-PCR reaction mixture (ice operation) into the PCR tube of step 5.4, mix well, centrifuge instantaneously, put into the PCR instrument to run the PCR reaction program of Table 6, take out the PCR tube and centrifuge instantaneously after the reaction.
TABLE 6PCR reaction procedure (Hot lid: 105 ℃ C.)
Post PCR purification
7.1 transfer PCR products to a new 1.5mL centrifuge tube, add 100 μ L of purified magnetic beads which have been balanced to room temperature, place on a vortex oscillator to mix thoroughly, after instantaneous centrifugation, stand for 5-10min, and the magnetic frame adsorbs to clarify.
7.2 carefully remove the supernatant by aspiration, add 200. mu.L of 75% ethanol and let stand for 1min until the liquid is clear. This step was repeated once.
7.3 carefully remove the supernatant, keep the centrifuge tube on the magnetic frame, open the tube lid, and dry it at room temperature until the surface of the magnetic beads is not reflective.
7.4 taking down the centrifuge tube, adding 32 mu L of elution buffer solution, placing on a vortex oscillator for fully and uniformly mixing, standing for 10min after instantaneous centrifugation, and absorbing 30 mu L to obtain a DNA library product.
8. Hybridization of probes
8.1 according to the concentration of the sample library, equally mixing the pre-PCR products into a new 1.5ml centrifuge tube, marking the total amount as 2 mug of the hybridization substrate, mixing the hybridization substrate and the hybridization substrate evenly, and then performing instantaneous centrifugation.
Note that: when equal amounts of pre-PCR products from N samples were mixed, the sampling volume (. mu.L) for each sample was 2000 ng/N/library concentration (ng/. mu.L), and N was 40 or less.
8.2 according to the concentration of the hybridization substrate in the step 8.1, taking 1.5 mu g of the hybridization substrate into a 1.5mL centrifuge tube, adding 44 mu L of hybridization reaction liquid, fully and uniformly mixing, carrying out instantaneous centrifugation, opening a slot shaped like a Chinese character 'mi' at the top of a cover, and concentrating the mixture to be dry powder in a vacuum dryer at the V-AQ mode of 60 ℃;
8.3 hybridization reaction mixtures were prepared in the amounts required for detection according to the ratios shown in Table 7, taking into account losses during preparation.
TABLE 7 hybridization reaction mixtures
| Name (R) | One reaction standard amount |
| xGen2XHybridizationBuffer | 8.5μL |
| xGenHybridizationBufferEnhancer | 2.7μL |
| xGenLockdownPanelorcustomprobes | 4μL |
| Nuclease-free water | 1.8μL |
| Total volume | 17μL |
Note: XGen2XHybridization buffer, if crystallized, was incubated at 65 ℃ with intermittent shaking until dissolved.
8.4 taking out the centrifuge tube in 8.2 and sealing, opening the cover after centrifugation, adding 17 mu L of hybridization reaction mixed solution, blowing, sucking, uniformly mixing, standing at room temperature for 5-10min, shaking, uniformly mixing, performing instantaneous centrifugation, transferring to a new 0.2ml PCR tube, and placing in a PCR instrument to run the PCR reaction program of the table 8.
TABLE 8 hybridization reaction procedure (Hot lid: 100 ℃ C.)
| Temperature of | Time of day |
| 95℃ | 30s |
| 65℃ | ≥12h |
| 65℃ | Hold |
9. Elution after hybridization
9.1 streptavidin magnetic beads room temperature to stand for 30min to balance to the room temperature, vortex and shake 15s mixing, 50 u L in new 0.2ml PCR tube.
9.2 Add 100 u L hybridization washing 1(xGen 1x BeadWashBuffer), gently blow and suck the mixture evenly for 10 times, place the mixture on a magnetic rack for about 1min until the mixture is clear, and suck and discard the supernatant.
9.3 repeat step 9.2 twice a total of 3 times.
9.4 get the step of 9.2 magnetic bead resuspension in 17 u L to 9.3 PCR tube, fully shake to heavy suspension, instantaneous centrifugation.
9.5 remove the hybridization reaction from the PCR instrument, add 17. mu.L of the resuspended beads from step 9.4, mix well with gentle vortex, and centrifuge instantaneously.
9.6 placing the mixture obtained in the step 9.5 in a PCR instrument, reacting for 45min at 65 ℃, covering the temperature with a hot cover at 70 ℃, mixing uniformly once every 10-12min by gentle vortex, and immediately carrying out hot elution after the reaction is finished.
9.7 Add 100. mu.L of the hybridization Wash 2 after warm bath (15min or more) to the reaction solution of step 9.6, blow and suck 10 times, do not generate bubbles as much as possible, put on the magnetic rack for 1min, and remove the supernatant.
9.8 remove the PCR tube on the magnetic frame, add 150 μ L of hybridization wash solution 5 through warm bath (15min or more), blow and suck 10 times, do not generate air bubbles as much as possible, incubate for 5min at 65 ℃, place on the magnetic frame for 1min, and suck and discard the supernatant.
9.9 repeat step 9.8 1 run for 2 times.
9.10 remove PCR tubes in step 9.9 Add 150. mu.L of hybridization Wash 2, vortex well to resuspend, incubate at room temperature for 2min, during which vortex 30s, rest 30s to ensure the mixture is homogeneous.
9.11 after the incubation, the mixture was placed on a magnetic frame for 1min by instantaneous centrifugation, and the supernatant was discarded by aspiration.
9.12 to the step 9.11 product add 150 u L hybridization washing solution 3, fully vortex to heavy suspension, room temperature incubation for 2min, during the period of vortex 30s, the rest of 30s, to ensure the mixture is uniform.
9.13 after incubation, placing on a magnetic frame for 1min by instantaneous centrifugation, and removing supernatant by suction.
9.14 to the 9.13 product add 150 u L hybridization washing solution 4, fully vortex to heavy suspension, room temperature incubation for 2min, in the period of vortex 30s, rest 30s, to ensure the mixture is uniform.
9.15 after incubation, placing the mixture on a magnetic frame for 1min by instantaneous centrifugation, sucking and discarding the supernatant, and replacing a new gun head to suck up the residual liquid.
And (3) taking down the PCR tube in the step 9.15, adding 20 mu L of nuclease-free water for resuspension of magnetic beads, uniformly mixing by blowing and sucking for 10 times, and performing instantaneous centrifugation.
Hybridization wash 1, hybridization wash 2, hybridization wash 3, hybridization wash 4, hybridization wash 5 were purchased from IDT, cat #: 1080584. the names of the five hybridization washes are as follows:
hybridization wash 1: xGen1 × BeadWashBuffer; hybridization wash 2: xGen1 × WashBuffer 1; hybridization wash 3: xGen1 × WashBuffer 2; hybridization wash 4: xGen1 × WashBuffer 3; hybridization wash 5: xGen1 × StringentWashBuffer.
Post-PCR amplification
10.1 Post-PCR reaction mixtures were prepared on ice according to the proportions of Table 9.
TABLE 9Post-PCR reaction mixtures
| Name of reagent | One reaction Standard quantity (. mu.L) |
| PCR reaction solution | 25 |
| PCR primer | 5 |
In Table 9, the PCR reaction solution was purchased from Shenzhen Huazhiyu science and technology Limited, AlphaHigh-FidelityReadyMix, cat No. 01K00901 MM.
The primers in table 9 are as follows:
an upstream primer: 5'-GAACGACATGGCTACGA-3', respectively;
a downstream primer: 5'-TGTGAGCCAAGGAGTTG-3' are provided.
10.2 Add 30. mu.L of the mixture of the LPost and PCR reactions to the PCR tube of step 9.15 (ice), mix well, centrifuge instantaneously, place in the PCR apparatus to run the PCR reaction program of Table 10, take out the PCR tube and centrifuge instantaneously after the reaction.
TABLE 10Post-PCR reaction procedure (Hot lid: 105 ℃ C.)
Post-PCR product purification
11.1 adding 75 μ L of purified magnetic beads into the product obtained in the step 10.2, shaking, mixing uniformly, standing for 5-10min, adsorbing by a magnetic rack until the mixture is clear, removing the supernatant, adding 125 μ L of 80% ethanol, incubating for 1min, removing the ethanol, washing repeatedly for 1 time, and air-drying at room temperature until the surfaces of the magnetic beads are not bright.
11.2 adding 22 mul elution buffer solution into the air-dried magnetic beads prepared in the step 11.1, shaking, mixing uniformly, standing for 5min, adding magnetic force for adsorption for 1-2min, carefully absorbing 20 mul eluent into a new centrifugal tube to obtain a purified product, and quantifying the DNA concentration by using a QubitdsDNAHSAssaykit and a matched instrument.
12. Automated analysis of sequencing and variation
The library is sequenced by adopting an MGI-SEQ2000 sequencer, the effective depth of a single sample is not less than 100x, and then mutation analysis is carried out on sequencing data by adopting mutation analysis software (various genetic disease gene carrier screening analysis software (V1), Huada Biotechnology (Wuhan) Co., Ltd.).
(II) results of detection of SMA-associated genetic variation in 418 samples
And (2) detecting 418 samples by referring to the method in the step (I), wherein the number of the probes in the kit is 1270, and the probes are specifically shown as SEQ ID No. 1-SEQ ID No.1270 in Table 1. The kit and a traditional qPCR method (a motor neuron survival gene 1(SMN1) exon deletion detection kit (a fluorescent quantitative PCR method) and Shanghai Wuchromashi medical research corporation) are used for detecting SMA-related SMN1 gene exon 7 deletion of 418 samples with known clinical results.
Results of detection of deletion variation of exon 7 of SMN1 gene related to SMA from Table 11418 samples
The detection results are shown in table 11, the samples with unqualified quality control and the samples in the gray area are processed according to the retest, and the SMA detection retest rate based on the kit is 1.4 percent and is obviously lower than the retest rate (9.6 percent) based on a qPCR method; and the sensitivity and specificity of SMA mutation detection based on the kit are superior to those of the traditional qPCR method. Therefore, compared with a qPCR method, the kit has better detection performance and is more suitable for screening and diagnosing the carriers of large SMA groups.
Example 2
Carrier screening of 6006 normal populations
The carrier screening was performed on 6006 normal populations using the same kit as in example 1, and the mutation detection protocol was as described in step (one) of example 1.
In a detection sample, the target regions of 5 genetic diseases have uniform depth and coverage, which shows that the probe has good capture uniformity, high targeting rate and no GC preference.
Of the 6006 normal specimens, 122 specimens were detected to carry pathogenic variation associated with hepatolenticular degeneration, 99 specimens to carry pathogenic variation associated with cblC type methylmalonemia, 87 specimens to carry pathogenic variation associated with SMA, and 217 specimens to carry pathogenic variation associated with thalassemia (. alpha. + β).
If the traditional method is adopted to realize the carrier screening of the 5 diseases, 3 methods are combined (qPCR method + sanger sequencing + Gap-PCR method), the process is complicated, the cost is high, and the flux is low. Therefore, compared with the traditional method, the kit can screen 5 diseases at one time, simplifies the detection process, improves the detection flux and reduces the detection cost.
Further, the invention is also suitable for screening the carriers of the 5 diseases. The invention is also suitable for screening the disease carriers of normal people.
Example 3
The probes were synthesized using two different probe synthesis methods. Synthesis scheme 1: each probe is independently synthesized and independently controlled in quality; synthesis scheme 2: all probes covered by the target area are mixed together for synthesis, and the length of the synthesized probe is 80-100 nt. The above probes were tested according to the method shown in example 1 using 350 samples, and the sequencing data is shown in table 12, and the repetition rate of the data (19%) in the captured sequencing data based on the probe of scheme 1 is significantly lower than the repetition rate of the captured sequencing data (51%) of the probe of scheme 2, resulting in the effective depth of the captured sequencing data (222X) of the probe in scheme 1 being significantly higher than the effective depth of the captured sequencing data (140X) of the probe of scheme 2 under the same data volume condition. Therefore, under the condition of obtaining the same effective depth, the data amount required by the scheme 1 probe capture sequencing is lower than that required by the scheme 2 probe capture sequencing, so that the cost of sample detection is reduced.
TABLE 12 Probe testing for different synthetic formats
| Evaluation index | Scheme 1 Probe | Scheme 2 Probe |
| Number of samples (n) | 350 | 350 |
| Data volume (G) | 1.1 | 1.2 |
| Capture efficiency (%) | 33 | 37 |
| Data repetition rate (%) | 19 | 51 |
| Effective depth (X) | 222 | 140 |
The uniformity of coverage of the capture region for the probes synthesized based on the two protocols is shown in FIG. 2, and the uniformity of coverage of the target region for the probes synthesized based on protocol 1 capture sequencing data is better than that of protocol 2.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (10)
1. A probe composition that specifically captures the causative genes of genetic disorders including α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, and methyl malonemia cblC type.
2. The probe composition of claim 1, wherein the length of each probe in the non-homologous region capture probe composition of the disease causing gene is the same;
and/or the length of each probe in the non-homologous region capture probe composition of the pathogenic gene is 120 nt;
and/or the length of the partial homologous region capture probe composition probe of the pathogenic genes of alpha-thalassemia, beta-thalassemia and spinal muscular atrophy is 110-120 nt;
and/or the fault tolerance of each probe in the probe composition is less than 7 nt;
and/or the coverage density of each probe in the probe composition is 1 x.
3. The probe composition of claim 1, wherein the target capture area of the probe composition comprises the following areas:
(a) the pathogenic genes related to alpha-thalassemia HBA1 and HBA 2;
(b) a pathogenic gene HBB related to beta-thalassemia;
(c) spinal muscular atrophy-related virulence gene SMN 1;
(d) pathogenic gene ATP7B related to hepatolenticular degeneration;
(e) the pathogenic gene MMACHC related to the methyl malonic acidemia cblC type;
and/or, for said region (a), aiming at full-length fragments of said pathogenic genes HBA1 and HBA2 and extending them by 50bp each upstream and downstream as final target capture region;
and/or, in the region (a), for-SEAAbsence, -alpha3.7Absence, -alpha4.2Deletion, selecting 1 SNP locus every 1kb in a deletion range as a target capture area;
and/or, regarding the region (b), aiming at the full-length fragment of the pathogenic gene HBB and extending the full-length fragment to the upstream and downstream by 50bp respectively to serve as a final target capture region;
and/or, in the region (b), for mainland type deletion, Taiwan type deletion, SEA-HPFH deletion, 1 SNP locus is selected as a target capture region every 1kb in the deletion range;
and/or, for said region (c), the whole genome region of said disease causing gene SMN1 is used as a target capture region;
and/or, for said region (d), using the exon region of the pathogenic gene ATP7B as the final target capture region;
and/or, for said region (e), using the exon region of the disease-causing gene MMACHC as the final target capture region.
4. The probe composition of claim 1, wherein the probe composition comprises at least one of 1270 nucleotide sequences that specifically capture the alpha-thalassemia-associated pathogenic genes HBA1 and HBA2, beta-thalassemia-associated pathogenic gene HBB, spinal muscular atrophy-associated pathogenic gene SMN1, hepatolenticular degeneration-associated pathogenic gene ATP7B, methylmalonemia cblC-type associated pathogenic gene MMACHC, or at least one of complements of the 1270 nucleotide sequences, respectively;
and/or the 1270 nucleotide sequence is shown as SEQ ID No. 1-SEQ ID No.1270 in the table 1;
and/or the probe composition has at least 80% sequence identity with at least one sequence of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or complementary sequences thereof;
and/or the probe composition comprises at least one sequence of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or a complementary sequence thereof;
and/or the probe composition comprises all of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or complementary sequences thereof.
5. A method for preparing a probe composition for screening carriers of pathogenic genes of genetic diseases is characterized by comprising the following steps:
a target capture region selection step of selecting respective target capture regions according to the target genes;
and designing probes according to the target capture region, wherein the lengths of the capture probes in the non-homologous regions of the pathogenic genes in the target capture region are the same, and the length of the capture probe in the partial homologous region of the pathogenic genes in the target capture region is 110-.
6. The method of claim 5, wherein each probe in the capture probe composition for the non-homologous region of the disease-causing gene has a length of 120 nt;
and/or, after designing the probes, synthesizing each probe separately;
and/or after each probe is independently synthesized, independently controlling the quality;
and/or, after designing the probe, synthesizing the probe at an equal concentration;
and/or, after the probes are synthesized in equal concentration, the probes are mixed in equal amount;
and/or the fault tolerance rate of each probe is less than 7 nt;
and/or, the coverage density of each probe is 1 x;
and/or, the genetic disease comprises at least one of α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, methylmalonic acidemia cblC type, preferably, the genetic disease comprises all diseases of α -thalassemia, β -thalassemia, spinal muscular atrophy, hepatolenticular degeneration, methylmalonic acidemia cblC type;
and/or the length of the partial homology region capture probe of the pathogenic genes of alpha-thalassemia, beta-thalassemia and spinal muscular atrophy is 110-120 nt;
and/or, the target capture area comprises at least one of:
(a) the pathogenic genes related to alpha-thalassemia HBA1 and HBA 2;
(b) a pathogenic gene HBB related to beta-thalassemia;
(c) spinal muscular atrophy-related virulence gene SMN 1;
(d) pathogenic gene ATP7B related to hepatolenticular degeneration;
(e) the pathogenic gene MMACHC related to the methyl malonic acidemia cblC type;
and/or the target capture area comprises all of the areas (a) - (e);
and/or, for the region (a), aiming at full-length fragments of the pathogenic genes HBA1 and HBA2 and extending the full-length fragments to 50bp respectively at the upstream and downstream to serve as target capture regions;
and/or, in the region (a), for-SEAAbsence, -alpha3.7Absence, -alpha4.2Deletion, selecting 1 SNP locus every 1kb in a deletion range as a target capture area;
and/or, regarding the region (b), aiming at the full-length fragment of the pathogenic gene HBB and extending the full-length fragment to the upstream and downstream by 50bp respectively to be used as a target capture region;
and/or, in the region (b), for mainland type deletion, Taiwan type deletion, SEA-HPFH deletion, 1 SNP locus is selected as a target capture region every 1kb in the deletion range;
and/or, for said region (c), the whole genome region of said disease causing gene SMN1 is used as a target capture region;
and/or, for said region (d), using the exon region of the pathogenic gene ATP7B as the final target capture region;
and/or, for said region (e), using the exon region of said disease-causing gene MMACHC as the final target capture region
And/or the probe composition comprises at least one of 1270 nucleotide sequences of a pathogenic gene HBA1 and HBA2 related to alpha-thalassemia, a pathogenic gene HBB related to beta-thalassemia, a pathogenic gene SMN1 related to spinal muscular atrophy, a pathogenic gene ATP7B related to hepatolenticular degeneration, a pathogenic gene MMACHC related to methyl malonatemia cblC type, or at least one of complements of the 1270 nucleotide sequences, respectively, specifically captured;
and/or the 1270 nucleotide sequence is shown as SEQ ID No. 1-SEQ ID No.1270 in the table 1;
and/or the probe composition has at least 80% sequence identity with at least one sequence of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or complementary sequences thereof;
and/or the probe composition comprises at least one sequence of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or a complementary sequence thereof;
and/or the probe composition comprises all of the sequences shown in SEQ ID No. 1-SEQ ID No.1270 or complementary sequences thereof.
7. A gene chip on which the probe composition according to any one of claims 1 to 4 or the probe composition prepared by the preparation method according to any one of claims 5 to 6 is immobilized.
8. A kit comprising the probe composition according to any one of claims 1 to 4, the probe composition prepared by the preparation method according to any one of claims 5 to 6, or the gene chip according to claim 7.
9. An assay device comprising the probe composition according to any one of claims 1 to 4, or the probe composition prepared by the preparation method according to any one of claims 5 to 6, or the gene chip according to claim 7, or the kit according to claim 8.
10. A sequencing method, comprising: capturing a target region by using the probe composition according to any one of claims 1 to 4, the probe composition prepared by the preparation method according to any one of claims 5 to 6, the gene chip according to claim 7, the kit according to claim 8, or the detection device according to claim 9;
and/or, the sequencing method further comprises: a step of genome DNA fragmentation and fragment double selection, a step of phosphorylation reaction and adaptor connection, a step of fragment enrichment, and then a step of sequencing is carried out by capturing a target region of the enriched fragment by using the probe composition of any one of claims 1 to 4, or the probe composition prepared by the preparation method of any one of claims 5 to 6, or the gene chip of claim 7, or the kit of claim 8, or the detection device of claim 9, and obtaining a sequencing result by high-throughput sequencing and automated analysis of variation;
and/or the double selection step of the genomic DNA fragments comprises:
a primary selection step, wherein magnetic beads are adopted to perform primary selection on the broken DNA fragments, and large fragments are removed to obtain primary selection products;
a second selection step, namely, performing secondary screening on the primary selection product by using the magnetic beads again;
and/or, in the initial selection, the ratio of the volume of the DNA to the volume of the added magnetic beads is 5: 3;
and/or, when two choices are selected, the ratio of the volume of the DNA to the volume of the added magnetic beads is 20: 3.
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| CN112359109A (en) * | 2020-11-26 | 2021-02-12 | 北京迈基诺基因科技股份有限公司 | Probe set and kit for detecting alpha thalassemia and beta thalassemia related pathogenic genes |
| CN113689914A (en) * | 2020-12-17 | 2021-11-23 | 武汉良培医学检验实验室有限公司 | Screening method and chip for single-gene genetic disease expansibility carrier |
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