Primer array for detection of mitochondrial dna haplotype in Han population and its preparation method and application
A technology for detecting primers and mitochondria, applied in the field of primer arrays, can solve the problems of complex procedures, cumbersome, heavy workload, etc., and achieve the effect of simple operation, high sensitivity and good specificity
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Embodiment 1
[0029] Example 1 Preparation of samples to be tested (using Genomic DNA Extraction Kit from Tiangen Company)
[0030] 1. Specimen Processing
[0031] Venous peripheral blood (a wide range of sources, can come from patients or research objects in various hospitals and scientific research units, and the venous peripheral blood of the present invention comes from the Second Affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army): get 300 μl of peripheral blood samples Add 900μl red blood cell lysate to a sterile 1.5ml EP tube, invert and mix well, and place at room temperature for 5 minutes, during which time invert several times. Centrifuge at 10,000 rpm for 2 minutes. Discard the supernatant, add 200 μl buffer GA to the pellet, and shake to mix.
[0032]2. Add 20 μl proteinase K and mix well.
[0033] 3. Add 200μl buffer GB, mix thoroughly by inverting, place at 70°C for 10-15 minutes, the solution becomes clear, and briefly ce...
Embodiment 2
[0041] Example 2 Design of specific PCR primers for Q-PCR
[0042] Human mtDNA is a double-stranded closed-loop molecule with 16,569 base pairs, which is responsible for encoding 2 rRNAs, 22 tRNAs, and 13 polypeptides involved in mitochondrial respiratory chain electron transport.
[0043] 1. Download the complete mitochondrial DNA Cambridge Sequence (rCRS) sequence from www.mitomap.org.
[0044] 2. Use Primer 5.0 software to design primers. Primer design principles: 1) Primers should be designed in the conserved region of the nucleic acid series and have specificity; 2) The length of the primer is generally between 15 and 30 bases; 3) The GC content of the primer is between 40% and 60%, and the Tm value is the highest. It is very close to 72°C; 4) The 3' end of the primer should avoid the third position of the codon; 5) A should not be selected at the 3' end of the primer, T is better; 6) The single strand of the amplification product cannot form a secondary structure; 7)...
Embodiment 3
[0047] Example 3 Design of detection and monitoring system
[0048] In order to ensure the reliability of the detection, a complete detection monitoring system is set up, including "yes" and "no" Q-PCR specific primers, positive controls, and negative controls. The Q-PCR specific primer sequence comes from the complete sequence of human mitochondrial DNA, and the known Han mitochondrial DNA haplotype genomic DNA (from the Second Affiliated Hospital of Third Military Medical University) was used as a positive control, containing only primers but without any The spotting solution of the DNA template is used as a negative control, the positive control is used to detect whether the Q-PCR fluorescence signal is correct, the negative control is used to detect whether the preparation of the primer array is normal, the positive and negative controls are processed in parallel with the sample, and are used to monitor the concentration of the sample to be tested. detection.
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