Molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof
A molecular marker, natural length technology, applied in the field of animal molecular genetics, can solve the problems of long time consumption, low efficiency of forced enzyme digestion, and complicated operation, and achieve the effect of high accuracy, simple primer design, and simple experimental operation.
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Embodiment 1
[0035] Obtaining Genomic DNA of Chinese Merino Sheep
[0036] The sheep ear tissues were collected and stored at -20°C for future use. The sheep genomic DNA was extracted by conventional phenol / chloroform method. The specific method is as follows:
[0037] (1) Take 5 g of ear tissue from Chinese Merino sheep (Xinjiang Army Reclamation type), remove the connective tissue, clean and disinfect the tissue block with 70% alcohol, put it in an Eppendorf tube, cut it with scissors, or grind it to pieces;
[0038] (2) After the alcohol in the Eppendorf tube is completely evaporated, add 700 μL of separation buffer, suspend the chopped tissue, add 5.0 μL of proteinase K (20 mg / mL), and act at 55 ° C for 8-12 hours until there are no tissue pieces;
[0039] (3) Take out the digested tissue fluid, add an equal amount of saturated phenol in the tissue fluid, mix for 10 minutes, centrifuge at 12,000 r / m at 4°C for 10 minutes;
[0040] (4) Take the supernatant, add the same amount of phe...
Embodiment 2
[0046] Obtaining the Digestion Product of Sheep TOMM70A Gene
[0047] According to the TOMM70A gene G163139235A site in the sheep genome, a pair of primers TOMM70AF and TOMM70AR were designed, and then the sheep genomic DNA was amplified by PCR to obtain the PCR amplification product, and then the PCR amplification product was digested with the endonuclease Bae I to obtain the enzyme cut product.
[0048] Such as figure 2 As shown, after the G163139235A site in the 7th intron region of TOMM70A gene is mutated, it can be completely recognized by BaeI endonuclease, so the primer sequence completely matches the template sequence, and no forced enzyme digestion is required.
[0049] The primer sequences are as follows:
[0050] TOMM70AF: 5'-ATGTGGCTTCTTGGAGTG-3'
[0051] TOMM70AR: 5'-CACAAATAAGCGCAGTTC-3'
[0052] The PCR amplification system is as follows:
[0053]
[0054] PCR amplification conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 53...
Embodiment 3
[0061] Determination of TOMM70A Genotype in Sheep
[0062] Use agarose gel with a concentration of 2% to 3% to carry out electrophoresis separation of the digested products in Example 1, and determine the genotype according to the results of the electrophoresis separation. The criteria for determination: (1) electrophoresis presents two bands, the size of which is 313bp and 152bp, when the G163139235A site in the 7th intron region of the sheep TOMM70A gene is not mutated, the PCR amplification product can be completely cut by Bae I enzyme, and it is named GG genotype; (2) Electrophoresis shows a band, If the size is 498bp, when the G163139235A site in the 7th intron region of the sheep TOMM70A gene is mutated, the PCR amplification product cannot be digested by Bae I, and it will be named as the AA genotype; (3) Electrophoresis shows three bands with a size of 498bp , 313bp and 152bp, the G163139235A site in the 7th intron region of the sheep TOMM70A gene is in a heterozygous ...
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