UCP2 gene mutation site of pig and detection method thereof
A genotype and polymorphic locus technology, applied in the field of molecular biology, can solve the relationship between fat deposition and energy metabolism, there has not been any research report yet.
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Embodiment 1
[0015] Example 1 The acquisition of UCP2 gene sequence and its polymorphic site
[0016] Select Landrace pigs, Yorkshire pigs, Duroc pigs, Min pigs, Jinhua pigs, Rongchang pigs, Tibetan pigs, and wild boar breeds to extract genomic DNA, which can be extracted from blood and tissues using conventional methods.
[0017] 1.1 PCR amplification
[0018] Primers were designed according to the reported mouse and human UCP2 gene sequences. The primer sequences are:
[0019] UCP2E1I1E2I2F 5′-ATGGTTGGATTCAAGGCCA-3′
[0020] UCP2E1I1E2I2R 5′-ATGCCAGCATCTGAGGGAGA-3′
[0021] UCP2E3I3E4F 5'-TGTGGTTCTCCCTCAGAT-3'
[0022] UCP2E3I3E4R 5'-CCATCTCCCCTGCCCCACCTGTC-3'
[0023] The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56-60°C for 30 s, extension at 72°C for 1 min, and 30 cycles; extension at 72°C for 7 min.
[0024] Purification, cloning and sequence determination of UCP2 amplification products
[0025] The PCR amplif...
Embodiment 2
[0033] Example 2 Genotype Detection of UCP2 Intron 4 C298T and C334T Mutations (PCR-SSCP)
[0034] Select DNA samples from 8 different breeds of pigs and design primers
[0035] U2I4S1F 5'-TCTGAAAGCAGACCCTCGGG-3'
[0036] U2I4S1R 5'-CGATGACGGTGGTGCAGAAG-3'
[0037] The PCR reaction conditions are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, and 30 cycles; extension at 72°C for 7 min, the amplification length is 160 bp, and the fragment includes UCP2 intron 4 Take a part of 1.5ul PCR product and place it in a PCR tube, add 5ul denaturing buffer, denature at 98°C for 10min, and rapidly ice-bath for 10min, and the denatured product undergoes 14% non-denaturing polyacrylamide gel electrophoresis, 140V, 14-16h , silver staining analysis, found that there are 6 genotypes, divided into AA, AB, BB, CC, BC and AC, the results are shown in Figure 1. Among them, the 298th base of AA type intron 4 (intron4)...
Embodiment 3
[0045] Example 3 Detection of UCP2 5th Intron 302-310 Deletion 9bp (Polypropylene Gel Electrophoresis)
[0046] Take pig DNA samples and design primers
[0047] UCP2intron5F 5'-GCTGCTTTCCTGCCTACTTG-3'
[0048] UCP2intron5R 5'-CGTTCCAGGACCCCAATCGG-3'
[0049] The PCR reaction conditions are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and 30 cycles; extension at 72°C for 7 minutes, amplification length: 224-233bp, the fragment includes UCP2 For the missing part of sub-5, take 2ul of the PCR product and place it in a PCR tube, add 5ul of Buffer, go through 14% polyacrylamide gel electrophoresis, 140V, 14-16h, and EB color analysis, and it is found that there is a deletion, as shown in Figure 2. Table 4 shows the distribution of CGGCGGTG G segment deletion genotypes of UCP2intron5 in different breeds of pigs.
[0050] Table 4 Detection of UCP2intron5 gene deletion in different br...
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