Primer combination for embryo preimplantation hereditary hearing loss gene detection, kit and application
A technology for hereditary deafness and preimplantation, applied in the field of molecular biology, can solve the problems of high risk of misdiagnosis, time-consuming, complicated operation, etc., so as to reduce the interference of results, improve the accuracy, and reduce the risk of misdiagnosis.
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[0059] Example 1 Analysis of pathogenic variants in family samples
[0060] 1. Sample preparation
[0061] The blood genomic DNA of both husband and wife and their affected offspring (proband) was extracted, the DNA concentration was measured by Qubit 2.0, and a certain amount was taken as the DNA sample to be tested.
[0062] 2. Target sequence capture and library construction
[0063] 1. Target Enrichment Capture
[0064] 1) After the DNA sample was vortexed and centrifuged, place it on an ice box, take two 0.2ml PCR tubes for each sample, and prepare the reaction system according to Table 3:
[0065] table 3
[0066] components
[0067] 2) After the preparation is completed, evenly distribute to two 0.2ml PCR tubes, 5μl in each tube. Add 5 μl of primer pool 1 to one tube, and 5 μl of primer pool 2 into the other tube, vortex to mix, and centrifuge briefly to concentrate the reaction system in the PCR tube at the bottom of the tube, put it into the PCR instrume...
Example Embodiment
[0101] Example 2 Pre-implantation detection of embryos
[0102] 1. Sample Preparation
[0103] Three to five cells from the trophectoderm of blastocyst-stage embryos were isolated as samples to be tested. The blastocyst stage embryos were sampled according to conventional embryo biopsy techniques, and the cells taken out were washed with 1×PBS (without Ca 2+ , Mg 2+ ) and washed 3 times, placed in a 0.2ml PCR tube with a volume not exceeding 2.5μl. Single-cell samples first use PicoPlex TM WGA kit for whole genome amplification.
[0104] 2. Target sequence capture and library construction
[0105] With embodiment 1.
[0106] The joints used for the 8 embryo samples to be tested G30218032601P01 to G30218032601P08 are SEQ ID NO.11 to SEQ ID NO.18 respectively.
[0107] 3. Fluorescent quantitative PCR to measure library concentration
[0108] With embodiment 1.
[0109] 4. On-machine sequencing and analysis of sequencing results
[0110] Dilute the library to 100pM, ta...
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