Primer group and kit for detecting PCCA gene
A technology of primer sets and kits, applied in biochemical equipment and methods, recombinant DNA technology, measurement/testing of microorganisms, etc., can solve the problems that children cannot be diagnosed
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Embodiment 1
[0078] The design of embodiment 1 primer
[0079] Using NM_000282.3 of the PCCA gene in the UCSC database as a reference sequence, in strict accordance with the principles of PCR primer design, 46 PCR amplification primers (a total of 23 pairs, Table 1) of exons 1-24 were designed using Primer Premier 5 software. The augmented region includes the exon region and the intron region greater than 50 bp at the junction of the exon and the intron, and the annealing temperature of all primers is designed to be almost the same.
[0080] Preliminarily ensure the accuracy of the primers through In-Silico PCR in the UCSC database;
[0081] Preliminarily ensure the specificity of the primers through Blat in the UCSC database;
[0082] Primer screening:
[0083] The down PCR reaction program was used to ensure that all primers had a good amplification effect, and agarose gel electrophoresis was performed on the amplification products of 23 pairs of primers. The results of electrophoresis ...
Embodiment 2
[0089] Embodiment 2 detects the method for PCCA gene
[0090] Proceed as follows:
[0091] (1) Use the 21 pairs of amplification primers obtained by screening to perform PCR amplification on the sample to be tested;
[0092] PCR amplification system (20μL): 2μL 2.5mM dNTP, 2.5μL containing Mg 2+ 10×buffer, 0.2μl 5U / μL Taq enzyme, 1μL 10μM forward primer, 1μL 10μM reverse primer, 1μL 50ng / μL template, add ddH 2 O filling;
[0093] PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 67°C for 30 s, extension at 72°C for 1 min, decreasing 0.5°C for each cycle, after 14 cycles, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, Extend at 72°C for 1 min, perform 30 cycles, extend at 72°C for 7 min, and finally hold at 4°C;
[0094] (2) After the amplification, the PCR amplification product is purified;
[0095] The purification system (10 μL) is: 1 μL 0.05 U / μL alkaline phosphatase, 0.5 μL 0.5 U / μL Exo I enz...
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