A kit for simultaneous analysis of 26 loci of human genome DNA by fluorescent labeling compound amplification and its use method and application
A technique for fluorescent labeling and compound amplification, applied in the field of five-color fluorescent label compound amplification system
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Embodiment 1
[0066] Example 1 A kit for simultaneous analysis of fluorescently labeled multiplexed amplification of 26 loci of human genomic DNA, consisting of the following:
[0067] components
volume
Reaction Mix
10.0 μL
Primers corresponding to 26 loci
5.0 μL
Hot start Taq enzyme (5U / μL)
0.5μL
wxya 2 o
Make up to 25.0 μL
[0068] The ReactionMix mentioned in it is MgCl2 7.5mM, Tris-HClbuffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg / mL.
[0069] The primers and primer concentrations corresponding to the 26 loci are:
[0070]
[0071] .
[0072] The 5' end of at least one of the primers corresponding to each locus is labeled with a fluorescent dye.
[0073] The fluorescent dye marker is 6-FAM, HEX, TAMRA or ROX.
[0074] The 26 loci are grouped, specifically: D3S1358, D13S317, D7S820, D16S539, D1S1656, and PentaE are the first group, and the fluorescent markers of the primers corresponding to this group of loci are those of ...
Embodiment 2
[0089] Application of 26-loci fluorescence-labeled multiple amplification test system for triplet paternity identification
[0090] 1. Collect blood samples in paternity testing cases: the samples in this experiment are provided by XX paternity testing agency. For DNA extraction, use the Chelex-100 method to extract genomic DNA from three whole blood samples: take 0.5-5 μl of whole blood and place it in a sterilized 1.5ml centrifuge tube, add 1ml of sdH2O to the tube, shake for a few seconds; place at room temperature for 10 minutes , shake for a few seconds, centrifuge at 12,000rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200 μl of 5% Chelex-100, shake for a few seconds; incubate at 56°C for 30 minutes, shake for a few seconds; Bath in boiling water for 10 minutes, shake for a few seconds; centrifuge at 12,000rpm for 5 minutes, and the supernatant is the extracted genomic DNA. Genomic DNA was e...
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