Micropterus salmoides sex specific SNP molecular marker primer and application thereof
A sex and primer pair technology, applied in the field of California perch sex-specific SNP molecular marker primers, can solve the problem of no California perch sex-specific molecular markers
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Embodiment 1
[0033] Example 1, the acquisition of California sea bass sex-specific SNP molecular markers
[0034] 1) During the breeding season, 24 females and 24 males of California perch were identified by dissecting and observing the gonads, and the tail fins were clipped and the whole genome DNA was extracted.
[0035] 2) 16 female California perch DNAs were mixed in equal amounts into a female mixed pool, and 16 male California perch DNAs were mixed in equal amounts into a male mixed pool.
[0036] A total of 18 samples from the female mixed pool, the male mixed pool, and the remaining 8 female and 8 male samples were subjected to whole-genome sequencing on the Illumina Hiseq X Ten platform. All the clean reads after filtering the original data were subjected to 60bp fragmentation electronic digestion starting from the 5' end of AC, AG, AT, GA, GC and GT six bases one by one, and the starting bases did not meet the requirements. rear displacement. The clean reads of the 8-tailed fem...
Embodiment 2
[0043] Embodiment 2, the application process of California perch sex-specific SNP molecular markers in identification
[0044] 1) Design the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2 according to the sequence shown in SEQ ID NO.3.
[0045] 2) Genomic DNA extraction (chelex100 boiling method)
[0046] Prepare 5% chelex100 solution; take 5mg of caudal fin tissue into a 0.2mL PCR tube, shake the chelex100 solution to evenly suspend the resin particles in the solution, and use a flaring tip to suck 150uL into the PCR tube containing the caudal fin tissue; put it into the PCR tube Add 5uL of 20mg / mL proteinase K solution, shake and mix well, place it on the PCR machine, set the conditions to heat the lid at 105°C, the module at 55°C, 40min; 98°C, 5min; take out immediately after the reaction, and vortex 3 times , 5s each time; put the PCR tube into the centrifuge and centrifuge at 4000rpm for 5min; the obtained supernatant is the DNA solution, which can be ...
Embodiment 3
[0052] Embodiment 3, California sea bass sex-specific SNP molecular marker stability detection
[0053] 1) The California perch body was randomly collected from the market, numbered 1-96, and the sex of the fish was determined by dissection, with 54 female fish and 42 male fish, and fin ray tissue samples were collected and stored in a 96-well plate for cryopreservation.
[0054] 2) Carry out HRM typing according to the application process provided in Example 2.
[0055] HRM typing results are as follows figure 1 and figure 2 shown. figure 1 The medium-dark curve represents the sample whose detected SNP locus is heterozygous, so it is judged as male (XY); the light-colored curve represents the sample whose detected SNP locus is homozygous, so it is judged as female (XX). figure 2 For the detection results of the 96-well plate, the two "S" marks in the figure are the standard curve reference automatically selected by the software, in which the dark blocks represent the sam...
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