Fluorescent multiplex amplification system of 37 Y-STR gene locuses and Y-Indel, kit and application thereof
A compound amplification system, y-indel technology, is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problem of cumulative individual identification and cumulative non-parent exclusion rate, which needs to be further improved, etc. problem, to achieve the effect of high cumulative individual recognition, high individual recognition rate, and strong species specificity
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Embodiment 1
[0046] Example 1 Selection of 37 Y chromosome STR loci and 1 Y-Indel, fluorescent label combination and primer design
[0047] In terms of locus selection, this product combines 20 core loci and 15 preferred loci from the Ministry of Public Security, adds a Y-Indel locus, and also contains a unique locus DYF404S1.
[0048] Divide 37 loci and 1 Y-Indel into five groups. After repeated experiments and verification, determine the optimal fluorescent dye combination scheme, Y-Indel, DYS393, DYS570, DYS19, DYS392, DYS549, Y-GATA-H4 , DYS444 is marked with FAM; DYS460, DYS458, DYS481, DYS635, DYS448, DYS533, DYS596 are marked with HEX; DYS456, DYS389I, DYS390, DYS389II, DYS438, DYS447 and DYS596 are marked with L552; DYS391, DYS439, DYS437, DYS385a / b DYS643, DYS518 are marked with LR600; DYS576, DYF404S1, DYF387S1, DYS627, DYS527a / b, DYS557 are marked with TET-592.
[0049] Design a pair of primers for each of the above loci, and perform amplification experiments with a single pair of pri...
Embodiment 2
[0059] Capillary electrophoresis detection of amplified products
[0060] Before detection, the product is denatured and mixed with the molecular weight internal standard, 1ul product + 0.5ul molecular weight internal standard + 8.5ul HIDI (deionized formamide), mix and centrifuge briefly, and immediately after denaturation at 95°C for 5 minutes, ice bath 10min, use ABI series genetic analyzer for electrophoresis detection, such as figure 1 . It can be seen from the figure that each locus can be typed accurately, with good specificity, no non-specific bands appear, and the peak height balance of each locus is also good.
Embodiment 3
[0062] Species specific experiments
[0063] Amplify the templates of pigs, cattle, sheep, and chickens, and detect the species specificity of 37 Y chromosome STR loci and 1 Y-Indel multiplex amplification system. The results are as follows figure 2 Shown. Through the amplification of other species, no bands were amplified, indicating that the 34 pairs of primers have good species specificity.
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