Bovine STR composite amplification detection kit, primer composition and application thereof
A primer composition and a technique for amplifying primers, applied in the field of biology
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Embodiment 1
[0110] Embodiment 1, the screening of bovine fluorescent STR composite amplification detection kit STR site and the design of primers
[0111] This embodiment provides a bovine fluorescent STR composite amplification detection kit, which includes a five-color fluorescent composite amplification primer system, through the research and optimization of the sequence composition characteristics of the conserved regions at both ends of the core repeat sequence of 16 bovine STR sites, Finally, 14 bovine STR loci (STR loci) were selected as target loci for primer design. The corresponding information of each STR locus (STR locus) is shown in Table 2:
[0112] Table 2 Information of bovine STR loci
[0113] site name Repeat sequence characteristics repeat sequence BM1818 simple (TG)n BM1824 simple (GT)n BM2113 simple (CA)n CSRM60 simple (AC)n CSSM66 simple (AC)n ETH3 compound (GT)nAC(GT) 6
ETH10 simple (AC...
Embodiment 2
[0144] Embodiment 2, multiplex amplification system primer concentration optimization
[0145] The optimization and establishment of the fluorescent STR multiplex amplification primer system is the core content of the development of the bovine fluorescent multiplex amplification kit. After completing the screening and design of single primers, it is necessary to mix each single primer and carry out multiple amplification debugging. Like single-site amplification, it is necessary to exclude primers that produce non-specificity and low amplification efficiency; finally, after initially determining After compound amplification primers for each locus, adjust the primer concentration. In the initial compound amplification, equimolar primers should be added to the reaction system, and then the concentration ratio of each pair of primers should be adjusted according to the reaction results; finally, the sequence and concentration of the primers should be determined through a large num...
Embodiment 3
[0149] Embodiment 3, bovine STR complex amplification buffer reagent system construction
[0150] The complex amplification reagent system determines the sensitivity, stability, amplification ability, amplification efficiency and many other key performance parameters of the reagents. First, based on the primer system prepared in Example 2, PCR buffer optimization and research were carried out. After a large number of PCR component and concentration optimization experiments, the influence of different components on the amplification performance of the system was explored, and a system optimization and evaluation method was established. On the basis of determining the components of each group, it is necessary to carry out the joint application test of each component, optimize the supporting cycle parameters, and finally establish the complex amplification buffer reagent system. The final reaction system is shown in Table 4, and the PCR cycle parameters are shown in Table 5. Show...
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