Human X chromosome 20 short tandem repeat multiplex amplification system and its application
A compound amplification system and short tandem repeat technology, applied in the field of biotechnology and biotechnology, can solve the problems of genetic polymorphism, individual identification and lack of accuracy of detection, etc.
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Embodiment 1
[0074] Example 1 Design of multiplex amplification system for 20 short tandem repeat sequences of human chromosome
[0075] 1. Primer Design
[0076] The primers are designed using software such as Prime Premier5 and NCBI Blast. When designing primers, it should be ensured that the Tm value of each primer is within the range of (60±2)°C, and the amplification efficiency is close to that of the amplification product of each pair of primers. different enough to be distinguished by capillary electrophoresis. After the design is completed, use software such as AutoDimer to analyze the interaction between the primer dimer and different primers. If there is an interaction that can produce non-specific products or dimers, it needs to be redesigned until the primer sequence that meets the requirements is obtained.
[0077] Select the human DNA template, use the above 20 pairs of primers to carry out single-plex amplification, place the amplified products on 1.5% agarose gel electroph...
Embodiment 2
[0085] Example 2 Genotyping detection of a certain sample
[0086] 1. Collection of blood samples (blood samples are donated by volunteers)
[0087] 2. DNA extraction
[0088] Genomic DNA was extracted using the Chelex-100 method (refer to "Forensic DNA Protocol". HumanaPress, 1998). Take 0.5-5 μl of anticoagulated whole blood or (1-3mm)*(2-5mm) blood spots in a 500 μl centrifuge tube, Shake and mix Chelex solution to fully suspend Chelex, add 195 μl Chelex-100 (5%) solution and 5 μl proteinase K (20mg / ml) to each tube, shake and mix, keep warm at 56 degrees Celsius for two hours or overnight, take out and shake for 2 minutes, boil water After heating in medium for 10 minutes, centrifuge at 13,000 rpm for 5 minutes, and carefully pipette 150 μl of the supernatant into a new centrifuge tube.
[0089] 3. Reaction system
[0090] Shake and mix each reaction reagent (buffer, primer mix, genomic DNA, etc.) and make a PCR reaction mixture in the following manner. The total volume...
Embodiment 3
[0102] Embodiment 3 is aimed at the optimization experiment of enzyme reaction buffer (2.5*Buffer D)
[0103] The concentration gradient of KCl in the enzyme reaction buffer is: 0.075M, 0.1M, 0.125M three concentration gradients. MgCl 2 The concentration gradient of the enzyme reaction buffer is: 1mM, 1.5mM, 2mM three concentration gradients. The concentration gradient of Brij58 in the enzyme reaction buffer is: 0.001mM, 0.0015mM, 0.002mM three concentration gradients. The concentration gradient of DTT in the enzyme reaction buffer is: 0.015M, 0.02M, 0.025M three concentration gradients. The concentration gradient of Betaine in the enzyme reaction buffer is: 0.05M, 0.1M, 0.15M three concentration gradients. The concentration gradient of DMSO in the enzyme reaction buffer is: 0.3%, 0.4%, 0.5% three concentration gradients. The concentration gradient of MR TaqⅡ in the enzyme reaction buffer is: 0.15U, 0.25U, 0.35U three concentration gradients. Orthogonal experiments with d...
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