Nucleic acid products for detecting copy number variations in the region of Chr12:q24.13~q24.21 and their applications

Abstract

The present application provides a nucleic acid product for detecting copy number variation in the region of Chr12:q24.13-q24.21 and its application. The nucleic acid product is selected from at least one pair among SEQ ID NO.1 to SEQ ID NO.256. By performing multiplex PCR using the above nucleic acid product, multiple SNP sites in the region of Chr12:q24.13-q24.21 and its upstream and downstream can be detected simultaneously before embryo implantation, so as to determine the genotype of the embryo and assist in determining whether the embryo has Holt-Oram syndrome.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and particularly relates to a nucleic acid product for detecting copy number variations in the region of Chr12:q24.13-q24.21 and its application. Background Art

[0002] With the continuous in-depth research on the genome and the rapid development of cell molecular detection technology, cases caused by chromosomal microdeletions or microduplications have been gradually discovered by the medical community. With the all-round development of precision medicine, the clinical significance of microdeletion and microduplication detection work has become more and more important. Selecting appropriate detection technology during detection can maximize the clinical promotion value of this technology. The region of Chr12:q24.13-q24.21 mainly contains two protein-coding genes (RBM19 and TBX5), among which the TBX5 gene is a pathogenic gene and a haploinsufficient gene included in the OMIM (Online Mendelian Inheritance in Man) database. Haploinsufficiency of the TBX5 gene and copy number variations in the region of Chr12:q24.13-q24.21 are related to Holt-Oram syndrome. Holt-Oram syndrome is an autosomal dominant inheritance, and its main clinical manifestations are upper limb abnormalities (such as carpal bone abnormalities), and most patients have cardiac abnormalities (atrial septal defect, ventricular septal defect, hypoplastic left heart), etc.; clinically, there are differences among individuals with Holt-Oram syndrome, and it is usually a pathogenic variation.

[0003] Currently, the genetic detection technologies for chromosomal microdeletions and microduplications mainly include: fluorescence in situ hybridization (FISH), chromosomal microarray chip technology (CMA), real-time fluorescence PCR technology, multiplex ligation-dependent probe amplification technology (MLPA), high-throughput sequencing technology (NGS), etc. However, there is currently no clear detection plan for preimplantation microdeletion and microduplication detection.

[0004] According to the latest guidelines proposed in the 2020 version of the European Society of Human Reproduction and Embryology (ESHRE), preimplantation genetic testing (PGT) refers to the technology of HLA typing or detecting genetic material abnormalities of DNA derived from oocytes (polar bodies) or embryos (cleavage stage or blastocyst stage), implanting disease-free embryos into the uterus for pregnancy, and giving birth to normal offspring. According to different detection purposes, PGT technology can be divided into three categories, including preimplantation genetic testing for aneuploidy (PGT-A), preimplantation genetic testing for monogenic diseases (PGT-M), and preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR).

[0005] Chromosomal aneuploidy, monogenic diseases, and copy number variation (CNV) are the main factors leading to embryonic abnormalities; however, current PGT detection methods generally can only detect CNVs above 3 Mb to 5 Mb, and there are certain limitations in detecting CNVs within 3 Mb. At present, for couples with known CNV syndromes, there is no very effective detection technology to accurately judge embryos before implantation. Summary of the Invention

[0006] Based on this, the present application provides a nucleic acid product for detecting microdeletions and microduplications in the Chr12:q24.13-q24.21 region before embryo implantation and its application.

[0007] According to the first aspect of the present application, a nucleic acid product for detecting copy number variation in the region of Chr12:q24.13-q24.21 is provided. The nucleic acid product includes one or more pairs of the following primer pairs: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, SEQ ID NO.59-60, SEQ ID NO.61-62, SEQ ID NO.63-64, SEQ ID NO.65-66, SEQ ID NO.67-68, SEQ ID NO.69-70, SEQ ID NO.71-72, SEQ ID NO.73-74, SEQ ID NO.75-76, SEQ ID NO.77-78, SEQ ID NO.79-80, SEQ ID NO.81-82, SEQ ID NO.83-84, SEQ ID NO.85-86, SEQ ID NO.87-88, SEQ ID NO.89-90, SEQ ID NO.91-92, SEQ ID NO.93-94, SEQ ID NO.95-96, SEQ ID NO.97-98, SEQ ID NO.99-100, SEQ ID NO.101-102, SEQ ID NO.103-104, SEQ ID NO.105-106, SEQ ID NO.107-108, SEQ ID NO.109-110, SEQ ID NO.111-112, SEQ ID NO.113-114, SEQ ID NO.115 to 116, SEQ ID NO. 117 to 118, SEQ ID NO. 119 to 120, SEQ ID NO. 121 to 122, SEQ ID NO. 123 to 124, SEQ ID NO. 125 to 126, SEQ ID NO. 127 to 128, SEQ ID NO. 129 to 130, SEQ ID NO. 131 to 132, SEQ ID NO. 133 to 134, SEQ ID NO. 135 to 136, SEQ ID NO. 137 to 138, SEQ ID NO. 139 to 140, SEQ ID NO. 141 to 142, SEQ ID NO. 143 to 144, SEQ ID NO. 145 to 146, SEQ ID NO. 147 to 148, SEQ ID NO. 149 to 150, SEQ ID NO. 151 to 152, SEQ ID NO. 153 to 154, SEQ ID NO. 155 to 156, SEQ ID NO. 157 to 158, SEQ ID NO. 159 to 160, SEQ ID NO. 161 to 162, SEQ ID NO. 163 to 164, SEQ ID NO. 165 to 166, SEQ ID NO. 167 to 168, SEQ ID NO. 169 to 170, SEQ ID NO. 171 to 172, SEQ ID NO. 173 to 174, SEQ ID NO. 175 to 176, SEQ ID NO. 177 to 178, SEQ ID NO. 179 to 180, SEQ ID NO. 181 to 182, SEQ ID NO. 183 to 184, SEQ ID NO. 185 to 186, SEQ ID NO. 187 to 188, SEQ ID NO. 189 to 190, SEQ ID NO. 191 to 192, SEQ ID NO. 193 to 194, SEQ ID NO. 195 to 196, SEQ ID NO. 197 to 198, SEQ ID NO. 199 to 200, SEQ ID NO. 201 to 202, SEQ ID NO. 203 to 204, SEQ ID NO. 205 to 206, SEQ ID NO. 207 to 208, SEQ ID NO. 209 to 210, SEQ ID NO. 211 to 212, SEQ ID NO. 213 to 214, SEQ ID NO. 215 to 216, SEQ ID NO. 217 to 218, SEQ ID NO. 219 to 220, SEQ ID NO. 221 to 222, SEQ ID NO. 223 to 224, SEQ ID NO. 225 to 226, SEQ ID NO.227 to 228, SEQ ID NO. 229 to 230, SEQ ID NO. 231 to 232, SEQ ID NO. 233 to 234, SEQ ID NO. 235 to 236, SEQ ID NO. 237 to 238, SEQ ID NO. 239 to 240, SEQ ID NO. 241 to 242, SEQ ID NO. 243 to 244, SEQ ID NO. 245 to 246, SEQ ID NO. 247 to 248, SEQ ID NO. 249 to 250, SEQ ID NO. 251 to 252, SEQ ID NO. 253 to 254, and SEQ ID NO. 255 to 256.

[0008] In one embodiment, the nucleic acid product includes primer pairs shown in SEQ ID NO. 1 to SEQ ID NO. 256.

[0009] According to the second aspect of the present application, there is provided the use of the above-mentioned nucleic acid product in the preparation of a kit for detecting chromosomal copy number variations.

[0010] According to the third aspect of the present application, there is provided a kit for detecting chromosomal copy number variations, characterized in that the kit includes the above-mentioned nucleic acid product.

[0011] In one embodiment, the kit further includes PCR reaction reagents.

[0012] In one embodiment, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, magnesium ions, and dNTPs.

[0013] In one embodiment, the kit further includes DNA extraction reagents.

[0014] According to the fourth aspect of the present application, a method for detecting the region of Chr12: q24.13 to q24.21 for non-diagnostic purposes includes the following steps:

[0015] Using the DNA of the sample to be tested as a template, performing PCR amplification using the above-mentioned nucleic acid product or the above-mentioned kit, and obtaining the sequence information of the SNP sites in the region of Chr12: q24.13 to q24.21 and its upstream and downstream of the sample to be tested according to the obtained amplification result;

[0016] Analyzing the sequence information, and determining whether there is a microdeletion and / or microduplication in the region of Chr12: q24.13 to q24.21 of the sample to be tested according to the obtained analysis result.

[0017] In one embodiment, the analysis method includes high-throughput sequencing.

[0018] In one embodiment, the DNA of the sample to be tested includes peripheral blood genomic DNA, semen DNA, buccal mucosal cell DNA, or whole-genome amplification products of cells.

[0019] Compared with the traditional technology, the present application has the following beneficial effects:

[0020] Using the nucleic acid product of the present application, multiplex PCR is performed on SNP sites inside and upstream and downstream of the Chr12:q24.13-q24.21 microdeletion / microduplication region, and then high-throughput sequencing is performed on the PCR amplification products. Combining pedigree information, the haplotype of mutant alleles is constructed, the genotype of the embryo is judged before embryo implantation, and finally the detection result of Holt-Oram syndrome in the offspring is determined; meanwhile, the nucleic acid product of the present application can detect 128 SNP sites simultaneously and detect CNVs smaller than 1 Mb.

[0021] In addition, the detection method of the present application can detect multiple samples simultaneously and has the advantages of low cost, strong versatility, and high accuracy. Detailed implementation manners

[0022] To make the above objects, features, and advantages of the present application more obvious and understandable, the specific implementation manners of the present application are described in detail. Many specific details are set forth in the following description to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this application can be obtained through the market or can be prepared by existing methods.

[0024] Term

[0025] Unless otherwise stated or there are contradictions, the terms or phrases used herein have the following meanings:

[0026] As used in this application, "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, mean greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0027] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0028] In this application, "reads" refers to the sequence fragments obtained by sequencing; "single nucleotide polymorphism (SNP)" refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level; "Haplotype" refers to a set of associated single nucleotide polymorphisms located in a specific region of a chromosome and tending to be inherited as a whole to the offspring, also known as haplotype or unit type; "sequencing depth" refers to the number of times the PCR amplification product is sequenced.

[0029] In the region of Chr12:q24.13 - q24.21 and its upstream and downstream, the following 128 SNP sites were screened out: Chr12:113066896, Chr12:113118535, Chr12:113238728, Chr12:113281488, Chr12:113335661, Chr12:113469275, Chr12:113736097, Chr12:113736121, Chr12:113787119, Chr12:113964164, Chr12:113984054, Chr12:113984089, Chr12:113996324, Chr12:114002967, Chr12:114040400, Chr12:114072329, Chr12:114089985, Chr12:114090057, Chr12:114092693, Chr12:114097168, Chr12:114097186, Chr12:114099314, Chr12:114130737, Chr12:114180110, Chr12:114182892, Chr12:114191494, Chr12:114202846, Chr12:114233531, Chr12:114238211, Chr12:114243033, Chr12:114243051, Chr12:114260240, Chr12:114281011, Chr12:114282678, Chr12:114287448, Chr12:114290903, Chr12:114334132, Chr12:114368054, Chr12:114390618, Chr12:114390643, Chr12:114455843, Chr12:114455875, Chr12:114469573, Chr12:114482116, Chr12:114482137, Chr12:114518101, Chr12:114538155, Chr12:114549194, Chr12:114556911, Chr12:114584058, Chr12:114621153, Chr12:114647734, Chr12:114669732, Chr12:114678318, Chr12:114678413, Chr12:114692372, Chr12:114714266, Chr12:114722082, Chr12:114725904Chr12:114725982, Chr12:114732927, Chr12:114751952, Chr12:114751994, Chr12:114849237, Chr12:114857694, Chr12:114887730, Chr12:114898346, Chr12:114904072, Chr12:114906528, Chr12:114917809, Chr12:114927076, Chr12:114929790, Chr12:114939989, Chr12:114948713, Chr12:115116352, Chr12:115129316, Chr12:115132047, Chr12:115195966, Chr12:115198520, Chr12:115206320, Chr12:115217113, Chr12:115261850, Chr12:115279610, Chr12:115279707, Chr12:115294800, Chr12:115300791, Chr12:115300817, Chr12:115351794, Chr12:115361364, Chr12:115367840, Chr12:115374658, Chr12:115398858, Chr12:115406458, Chr12:115510669, Chr12:115527101, Chr12:115527103, Chr12:115535403, Chr12:115541637, Chr12:115605004, Chr12:115619542, Chr12:115623600, Chr12:115623689, Chr12:115711483, Chr12:115715832, Chr12:115735505, Chr12:115762543, Chr12:115773462, Chr12:115783539, Chr12:115787973, Chr12:115792584, Chr12:115808685, Chr12:115945722, Chr12:115945782, Chr12:116074464, Chr12:116094969, Chr12:116167464, Chr12:116167524, Chr12:116167580, Chr12:116242225, Chr12:116242271, Chr12:116278292Chr12: 116278388, Chr12: 116299872, Chr12: 116312828, Chr12: 116442606, Chr12: 116480329, Chr12: 116762091 and Chr12: 116790821.

[0030] Based on the above SNP loci, some embodiments of the present application provide a nucleic acid product for detecting copy number variations in the region of Chr12:q24.13-q24.21, including one or more pairs of the following primer pairs: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, SEQ ID NO.59-60, SEQ ID NO.61-62, SEQ ID NO.63-64, SEQ ID NO.65-66, SEQ ID NO.67-68, SEQ ID NO.69-70, SEQ ID NO.71-72, SEQ ID NO.73-74, SEQ ID NO.75-76, SEQ ID NO.77-78, SEQ ID NO.79-80, SEQ ID NO.81-82, SEQ ID NO.83-84, SEQ ID NO.85-86, SEQ ID NO.87-88, SEQ ID NO.89-90, SEQ ID NO.91-92, SEQ ID NO.93-94, SEQ ID NO.95-96, SEQ ID NO.97-98, SEQ ID NO.99-100, SEQ ID NO.101-102, SEQ ID NO.103-104, SEQ ID NO.105-106, SEQ ID NO.107-108, SEQ ID NO.109-110, SEQ ID NO.111-112, SEQ ID NO.113-114, SEQ ID NO.115 - 116, SEQ ID NO. 117 - 118, SEQ ID NO. 119 - 120, SEQ ID NO. 121 - 122, SEQ ID NO. 123 - 124, SEQ ID NO. 125 - 126, SEQ ID NO. 127 - 128, SEQ ID NO. 129 - 130, SEQ ID NO. 131 - 132, SEQ ID NO. 133 - 134, SEQ ID NO. 135 - 136, SEQ ID NO. 137 - 138, SEQ ID NO. 139 - 140, SEQ ID NO. 141 - 142, SEQ ID NO. 143 - 144, SEQ ID NO. 145 - 146, SEQ ID NO. 147 - 148, SEQ ID NO. 149 - 150, SEQ ID NO. 151 - 152, SEQ ID NO. 153 - 154, SEQ ID NO. 155 - 156, SEQ ID NO. 157 - 158, SEQ ID NO. 159 - 160, SEQ ID NO. 161 - 162, SEQ ID NO. 163 - 164, SEQ ID NO. 165 - 166, SEQ ID NO. 167 - 168, SEQ ID NO. 169 - 170, SEQ ID NO. 171 - 172, SEQ ID NO. 173 - 174, SEQ ID NO. 175 - 176, SEQ ID NO. 177 - 178, SEQ ID NO. 179 - 180, SEQ ID NO. 181 - 182, SEQ ID NO. 183 - 184, SEQ ID NO. 185 - 186, SEQ ID NO. 187 - 188, SEQ ID NO. 189 - 190, SEQ ID NO. 191 - 192, SEQ ID NO. 193 - 194, SEQ ID NO. 195 - 196, SEQ ID NO. 197 - 198, SEQ ID NO. 199 - 200, SEQ ID NO. 201 - 202, SEQ ID NO. 203 - 204, SEQ ID NO. 205 - 206, SEQ ID NO. 207 - 208, SEQ ID NO. 209 - 210, SEQ ID NO. 211 - 212, SEQ ID NO. 213 - 214, SEQ ID NO. 215 - 216, SEQ ID NO. 217 - 218, SEQ ID NO. 219 - 220, SEQ ID NO. 221 - 222, SEQ ID NO. 223 - 224, SEQ ID NO.225 - 226, SEQ ID NO.227 - 228, SEQ ID NO.229 - 230, SEQ ID NO.231 - 232, SEQ ID NO.233 - 234, SEQ ID NO.235 - 236, SEQ ID NO.237 - 238, SEQ ID NO.239 - 240, SEQ ID NO.241 - 242, SEQ ID NO.243 - 244, SEQ ID NO.245 - 246, SEQ ID NO.247 - 248, SEQ ID NO.249 - 250, SEQ ID NO.251 - 252, SEQ ID NO.253 - 254, and SEQ ID NO.255 - 256.

[0031] The nucleic acid products of the embodiments of the present application include 1, 2, 3, 4, 5,..., 120, 121, 122, 123, 124, 125, 126, 127, 128 pairs of the above primer pairs.

[0032] It can be understood that by detecting chromosomal copy number variations, it is possible to understand whether there are microdeletions and / or microduplications in the chromosomes. Detecting the copy number variation in the region of Chr12:q24.13 - q24.21 can assist in confirming whether the pre - implantation embryo carries Holt - Oram syndrome.

[0033] In some specific examples, the above primer pairs have similar annealing temperatures, and the sizes of the PCR product fragments are all in the range of 125bp - 275bp.

[0034] In some of these embodiments, the above nucleic acid products include the primer pairs shown in SEQ ID NO.1 - SEQ ID NO.256, and the primer information is shown in Table 1.

[0035] Table 1 SNP Loci and Primer Sequence Table

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] Some other embodiments of the present application provide an application of the above nucleic acid product in preparing a kit for detecting chromosomal copy number variation.

[0043] Some other embodiments of the present application further provide a kit comprising the above nucleic acid product.

[0044] In some specific examples, the kit further comprises PCR reaction reagents.

[0045] In some specific examples, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, magnesium ions, and dNTPs.

[0046] In some specific examples, the kit further comprises DNA extraction reagents.

[0047] It can be understood that in some other specific examples, the above kit may not include any of the PCR reaction reagents and DNA extraction reagents, and the reagents not included can be reasonably obtained from the outside.

[0048] Using the kit of the present application to detect chromosomal copy number variation can simultaneously detect the sequence information of multiple SNP sites and multiple samples, and the detection result has high accuracy.

[0049] Some other embodiments of the present application further provide a method for detecting the region of Chr12:q24.13-q24.21 for non-diagnostic purposes, comprising step S10 and step S20:

[0050] Step S10: Using the DNA of the sample to be tested as a template, performing PCR amplification with the above nucleic acid product or kit, and obtaining the sequence information of the SNP sites in the region of Chr12:q24.13-q24.21 and its upstream and downstream of the sample to be tested according to the obtained amplification result.

[0051] Step S20: Analyzing the sequence information, and determining whether there is microdeletion and / or microduplication in the region of Chr12:q24.13-q24.21 of the sample to be tested according to the obtained analysis result.

[0052] In some specific examples, in step S20, the method for analyzing the sequence information includes high-throughput sequencing. It can be understood that the method for sequence analysis is not limited to this, and other existing methods can also be used for analysis. Optionally, the genomic reference sequence can be from a public database, such as the human genomic sequence can be the human genomic reference sequences hg19, hg38, etc. in the NCBI or UCSC database.

[0053] In some specific examples, step S20 further comprises: aligning the sequence information with the DNA sequences of both parents and analyzing the haplotype of the embryo.

[0054] In some specific examples, step S20 further includes: aligning the sequence information with the human genome reference sequence, and analyzing the SNP coverage multiple and genotype.

[0055] In some specific examples, the DNA of the sample to be tested includes peripheral blood genomic DNA, semen DNA, oral mucosal cell DNA, or whole-genome amplification product of cells.

[0056] It can be understood that the detection object of this method is an embryo that has not been implanted into the uterus, not a living human body or animal body, and its detection result does not involve the disease diagnosis results of both of its parents. Therefore, it does not belong to the method for diagnosing and treating diseases.

[0057] The above method for detecting the region of Chr12:q24.13-q24.21 has the advantages of strong generality, high accuracy, fast and efficient, etc.; by detecting the SNP site information in the region of Chr12:q24.13-q24.21, microdeletions and / or microduplications with a chromosomal region less than 1 Mb can be identified before embryo implantation, and the genotype of the embryo can be further analyzed; it provides a new idea for genetic testing before embryo implantation.

[0058] Hereinafter, the present application will be further described in conjunction with specific examples and comparative examples, but it should not be construed as a limitation to the protection scope of the present application.

[0059] Example 1: Screening of SNP Sites and Primer Design

[0060] (1) Screening of SNP sites: For the microdeletion / microduplication region of Chr12:q24.13-q24.21 and its upstream and downstream, high-frequency SNP sites are selected from the 1000 Genomes Project database (http: / / www.ncbi.nlm.nih.gov / variation / tools / 1000genomes / ); high-frequency SNP sites with a minor allele frequency greater than 0.2 are selected; SNP sites with a polyN (polynucleotide) and a GC content > 70% in the 50 bp sequence upstream and downstream of the site are removed; SNP sites with multiple alignment positions in the 50 bp sequence upstream and downstream aligned to the human genome hg19 are removed (i.e., SNP sites with high homology are removed); SNP sites within the gene and within 1 Mb upstream and downstream of the gene (or mutation site) are preferentially selected. If the number of SNPs within 1 Mb is small, the range can be appropriately expanded, such as 2 Mb upstream and downstream.

[0061] A total of 128 SNP sites in the microdeletion / microduplication region of Chr12:q24.13-q24.21 and its upstream and downstream are screened according to the above method.

[0062] (2) Primer design: Specific primers were designed for the 128 SNP sites screened in step (1) respectively (online platform https: / / www.ampliseq.com / ). The primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.256. These primers have similar annealing temperatures, and the fragment sizes of the PCR products are all in the range of 125bp to 275bp.

[0063] Example 2

[0064] A couple once had a fetus with congenital heart disease and then had an induced abortion. The chromosomal copy number variation (CNV) test report indicated a 0.80 Mb deletion in the region of Chr12:q24.13 - q24.21 (seq[hg19]12q24.13 - q24.21(114200001_115000000)×1), which was inherited from the male. The couple applied for preimplantation genetic testing (PGT) for the above pathogenic mutation to have a healthy offspring. Through PGT-assisted pregnancy, 6 embryos were obtained. When the embryos developed to the blastocyst stage, trophoblast cell biopsy was performed. After the biopsy cells were amplified by whole genome amplification, the haplotypes of the embryos were further determined. Among the 6 embryos, 3 were normal and 3 carried the deletion mutation in the region of Chr12:q24.13 - q24.21. The couple transplanted a normal embryo. Prenatal ultrasound diagnosis and chromosomal copy number variation (CNV) test were performed at 16 weeks of pregnancy. The B-ultrasound showed that the fetus developed normally, and the CNV test indicated that the fetal chromosomal copy number was normal, which was consistent with the PGT result. Subsequently, the pregnancy was successful and a healthy baby was born.

[0065] (1) Multiplex PCR amplification: The 128 pairs of primers synthesized in Example 1 were mixed into a single PCR reaction tube, and a 128-plex reaction was performed on the sample to amplify the SNP sites within and upstream and downstream of the Chr12:q24.13 - q24.21 microdeletion / microduplication region of the tested sample.

[0066] (2) Library construction and sequencing: The whole genome amplification product of the biopsy cells was constructed into a library according to the Illumina standard library construction process, and sequenced with Miseq. When the DNA molecules to be tested come from multiple test samples, different tag sequences (barcodes) can be added to each sample for differentiation, so as to achieve simultaneous sequencing of multiple samples.

[0067] (3) Sequencing analysis: Sequence alignment was performed through BWA (Burrow-Wheeler-Aligner), and the reads were aligned with the reference genome sequence to obtain the positions of the reads on the reference genome.

[0068] (4) Data analysis: The original data generated by the Illumina sequencer was processed using the trimmomatic software to remove adapter sequences, aligned to the human hg19 reference genome using the BWA software, and finally the haplotype SNP coverage multiple and genotype were analyzed; the detection results are shown in Table 2.

[0069] Table 2 Summary of Detection Results

[0070] Number Name Haplotype Pathogenic Gene Testing (PGT-M) K13467 Female M0 / M1 Normal V2206 Male F0 / F1 Pathogenic V2207 Induced Abortion Fetus M0 / F0 Pathogenic P1922-ZHJ1 ZHJ1 M1 / F1 Normal P1922-ZHJ2 ZHJ2 M1 / F1 Normal P1922-ZHJ3 ZHJ3 M0 / F1 Normal P1922-ZHJ4 ZHJ4 M1 / F0 Pathogenic P1922-ZHJ5 ZHJ5 M0 / F0 Pathogenic P1922-ZHJ7 ZHJ7 M0 / F0 Pathogenic

[0071] Note: M0 and M1 represent the normal chromosomes of the female; F0 represents the risk chromosome of the male, and F1 represents the normal chromosome of the male.

[0072] The quality control results are shown in Table 3. It can be seen that the average sequencing depth is above 100×, the 30× coverage is above 70%, and the 100× coverage is above 50%, indicating that the quality control is qualified.

[0073] Table 3 High-throughput Sequencing Quality Control

[0074]

[0075] The statistics of valid SNP sites are shown in Table 4. It can be seen that there are more than 2 heterozygous SNP sites within 1M on both sides of the Chr12:q24.13-q24.21 region for both the male and female, meeting the requirements of the "Practice Guidelines for Amplification-based PGT" (2020 edition) of the European Society of Human Reproduction and Embryology (ESHRE).

[0076] Table 4 Statistics of SNP Sites in Target Genes

[0077]

[0078] The SNP haplotypes of some selected embryos are shown in Table 5. Among them, F0 represents the risk chromosome of the male, and F1 represents the normal chromosome of the male; M0 represents the normal chromosome of the female, and M1 represents the normal chromosome of the female. Among them, sites 1-40 are all within the pathogenic sites; sites 10-15 are within the Chr12:q24.13-q24.21 microdeletion / microduplication region. Sites 11-25, 27, 31, 36-38 are valid sites for the female; sites 1-10, 26, 28-30, 32-35, 39-40 are valid sites for the male.

[0079] Table 5 SNP Haplotypes

[0080]

[0081]

[0082] Note: "?" in the above table represents that the site was not detected.

[0083] The above results indicate that, based on the high-throughput sequencing technology, the nucleic acid products of the present application can detect multiple SNP sites in the region of Chr12:q24.13-q24.21 and its upstream and downstream in the sample, and further determine whether the embryo is a carrier of the pathogenic gene of Holt-Oram syndrome by analysis and comparison.

[0084] Example 3

[0085] Using the method of Example 2, 602 samples were detected. The results showed that there were multiple effective SNP sites in the region of Chr12:q24.13-q24.21 and its upstream and downstream in the samples, which could provide sufficient genetic marker information to judge the risk signal, indicating that the primer pair of the present application has high generality and accuracy in detecting copy number variation in the region of Chr12:q24.13-q24.21.

[0086] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0087] The above-described embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A nucleic acid product for detecting copy number variation in the region of Chr12:q24.13 to q24.21, characterized in that, The nucleic acid product includes the primer pairs shown in SEQ ID NO.1 to SEQ ID NO.

256.

2. Use of the nucleic acid product according to claim 1 in the preparation of a kit for detecting chromosomal copy number variations.

3. A kit for detecting chromosomal copy number variations, characterized in that, The kit includes the nucleic acid product according to claim 1.

4. The kit for detecting chromosomal copy number variations according to claim 3, wherein, The kit further includes PCR reaction reagents.

5. The kit for detecting chromosomal copy number variations according to claim 4, characterized in that, The PCR reaction reagents include one or more of a PCR buffer, a DNA polymerase, magnesium ions, and dNTPs.

6. The kit for detecting chromosomal copy number variations according to any one of claims 3 to 5, characterized in that, The kit further includes DNA extraction reagents.

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