str type standard substance
A technology for typing reference materials and reference materials, applied in the field of forensic DNA testing and analysis, which can solve the problems of heavy workload, high cost, and difficulty in obtaining
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Embodiment 1、1
[0031]Embodiment 1, the acquisition of 19 locus alleles
[0032] 1. Locus CSF1P0 alleles (N=12 (TAGA) 12 ) of the acquisition
[0033] 1. Design of primers
[0034] On the basis of determining the flanking sequence of the CSF1P0 gene locus, the primers were designed as follows: Forward primer: TGGCAGAAGCCGGAGGTAA; Reverse primer: CCGATGAGCTGCTGCCTTG
[0035] 2. PCR amplification
[0036] Using the above primer pairs, PCR amplification was carried out using the extracted human saliva genomic DNA as a template. The PCR system and reaction parameters are as follows:
[0037] a. PCR amplification system (10 μL):
[0038]
[0039] b. PCR thermal cycle parameters:
[0040] 95℃11min; 94℃1min, 60℃1min, 72℃1min, 30cycles; 60℃60min; 25℃Hold. (The annealing temperature varies according to the Tm value of different loci.)
[0041] After electrophoresis, recovery, and purification, the PCR product fragments were directly inserted into the carrier T vector (purchased from Promega...
Embodiment 2
[0164] The preparation of the standard substance of embodiment 2, STR typing
[0165] The 31 recombinant cloning vectors prepared in Step 1 to Step 19 in Example 1 were gradually mixed. With the increase of allelic DNA fragments, due to the competition mechanism inside the DNA molecule, there was a certain degree of relative balance control of each allelic fragment. It is necessary to continuously adjust the recombinant plasmids with different allelic DNA fragments to the optimal ratio, and finally establish the relative balance among multiple DNA fragments through repeated experiments, and finally form a DNA standard substance that meets the requirements. Among them: the recombinant cloning vector V-CSF1 among the 31 recombinant cloning vectors: the recombinant cloning vector V-1338-1: the recombinant cloning vector V-1338-2: the recombinant cloning vector V-1358-1: the recombinant cloning vector V-1358- 2: Recombinant cloning vector V-818: Recombinant cloning vector V-1043-1...
Embodiment 3
[0167] Embodiment 3, verification of reference material
[0168] The amplification system in the amplification experiment from step 1 to step 4 is 10ul.
[0169] 1. Typer TM 15 kit amplification
[0170] Using the standard substance actually prepared in Example 2 as a DNA template, Typer TM 15 Fluorescence multiplex amplification kit (Ministry of Public Security Evidence Identification Center) was used for amplification according to the operating instructions. Its DNA test profile is as follows figure 1 As shown, the spectrum is clear and the peak shape is sharp, which is consistent with the preset results.
[0171] 2. Identifier TM kit amplification
[0172] Using the standard substance actually prepared in Example 2 as a DNA template, the Identifilier TM Fluorescence multiplex amplification kit (ABI Company, USA) was used to amplify according to the operating instructions. The amplification parameters were set as 95°C, 5min; 94°C for 1min, 59°C for 1min, 72°C for 1mi...
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