Kits for preimplantation genetic diagnosis and prenatal diagnosis of dmd
A preimplantation embryo and prenatal diagnosis technology, applied in the field of preimplantation embryo genetic diagnosis and prenatal diagnosis kits, can solve the problem of unaccounted for amplification preference of genomic regions, increased misreading of downstream analysis, and difficult detection of SNP In order to achieve the effect of improving efficiency and stability, low sequencing depth, and improving detection specificity and accuracy
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Embodiment 1
[0072] Embodiment 1: Design and preparation of the probe set of the present invention
[0073] 1. Screening of DMD gene-linked SNP loci
[0074] Based on the low-depth sequencing data of MALBAC amplification products of healthy embryo cells that have been sequenced and genetically abnormal embryos, 199 DMD genes in the upstream and downstream 2M regions were screened out according to the following steps. Linked SNP loci. The specific screening process and criteria are:
[0075] 1) For each existing sequencing sample, after normalizing the read coverage of the coverage site according to the sample sequencing depth, select the site whose coverage exceeds the ideal threshold (that is, the number of coverage is at least 3 times);
[0076] 2) Select sites that appear in more than 80% of all samples (in this case, a total of 24 embryos were selected from MALBAC amplification and pre-sequencing data), and merge adjacent sites, and the resulting region is determined to be a small nu...
Embodiment 2
[0086] two. Embodiment 2: Composition, preparation and use of the kit of the present invention
[0087] The detection kit for preimplantation genetic diagnosis and prenatal diagnosis of DMD gene described in this embodiment is a kit for molecular genetic detection by simultaneously detecting the mutation of DMD gene and its linked SNP site.
[0088] The kit contains the following components: the probe set obtained in Example 1, the DMD gene capture probe set (refer to CN 106834507 A), buffer HY, buffer BL, library enrichment binding buffer, washing buffer Solution WB1, washing buffer WB3, buffer NE, PCR reaction solution, product purification eluate. The specific composition is shown in Table 1. Table 1
[0089]
[0090] The using method of described test kit is:
[0091] Sample library preparation:
[0092] a) Ultrasonic fragmentation: the initial amount (the product of cellular genomic DNA amplified by MALBAC) is 3 μg, diluted to 30 ng / μL with 1× lowTE Buffer. Covar...
Embodiment 3
[0127] Embodiment 3: verification of the use effect of the kit of the present invention
[0128]The present invention will be described more comprehensively through specific embodiments below. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention.
[0129] The child in family 1 suffered from Duchenne muscular dystrophy (DMD, Exon31-43 deletion), X-linked recessive inheritance. The woman is a carrier (DMD, Exon31-43 heterozygous deletion), and the man is normal. See the specific genetic map figure 1
[0130] figure 2 It is the electropherogram of exon deletion of DMD gene in an example family. The order of exon loading is: MALBAC1, MALBAC2, MALBAC3, mother (carrier), father, progenitor (DMD Exon31-43 deletion), and the results show that the progenitor carries the homozygous deletion mutation of DMD gene exon31-43.
[0131] 1. Take human emb...
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