Primers, primer combinations, kits and their applications
A kit and primer set technology, applied in the direction of protozoa, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems affecting the sensitivity and specificity of amplification
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Embodiment 115
[0181] Example 1 Construction of 15 mutant plasmids
[0182] (1) Amplify the NPM1 fragment containing the insertion mutation: in such as Figure 5 Design primers at both ends of the shown insert fragments, design bidirectional primers containing deletion mutation fragments at the mutation site, and use bridge PCR to construct NPM1 fragments containing insertion mutations;
[0183] (2) Ligation and transformation reactions: use a commercial kit to connect the NPM1 mutant fragment and the PMD19-T vector and transform it into DH5α Escherichia coli;
[0184] (3) Pick positive clones and preserve species: Spread Escherichia coli on LB agarose medium containing ampicillin and culture overnight at 37°C, pick a single colony, and use the two-terminal specificity markers in the insert to select a part of them. Amplify with primers, and the cloned colonies that can amplify the target band size are sent to be sequenced and confirmed as positive, and then stored with 25% glycerol at -20°...
Embodiment 2
[0187] Embodiment 2 utilizes each primer real-time PCR to detect the specificity of each mutant separately
[0188] grouping:
[0189] Experimental group: 15 mutated plasmids constructed in Example 1;
[0190] The negative control is wild-type NPM1; the blank control is water.
[0191] (1) Plasmid dilution: Dilute wild-type (WT), typeA, typeB, typeD, typeDD1, typeE mutant NPM1 plasmids to 2.7x10 5 copies / uL.
[0192](2) Real-time PCR reaction: Prepare reaction system in PCR tube: 10 μl of commercial Taq enzyme PCR Mix (2x), 0.8 μl of primer X, 10.8 μl of primer, 10.5 μl of probe, mutant type diluted in (1) Plasmid / (1) diluted wild-type plasmid / water 1μl, cDNA extracted from HL60 cell line 1uL, make up to 20μl with sterilized deionized water;
[0193] Wherein, primer X is the corresponding primer (SEQ ID No.2~6) when detecting different mutant plasmids, the concentration is 4~8pmol / μl, and the concentration of primer 1 (SEQ ID No.7) is 4~8pmol / μl, The concentration of prob...
Embodiment 3
[0204] Embodiment 3 utilizes mixed primer to carry out real-time PCR detection to fifteen kinds of mutants
[0205] grouping:
[0206] Experimental group: 15 mutated plasmids constructed in Example 1;
[0207] The negative control is wild-type NPM1; the blank control is water.
[0208] (1) Plasmid dilution: Dilute wild-type (WT), typeA, typeB, typeD, typeDD1, typeE, typeF, typeG, typeH, typeI, typeJ, typeK, typeL, typeM, typeN, typeO mutant NPM1 plasmids to 5 Gradients: 2.7x10 5 copies / uL, 2.7x10 4 copies / uL, 2.7x10 3 copies / uL, 2.7x10 2 copies / uL, 2.7x10 2 copies / uL, 2.7x10 1 copies / uL.
[0209] (2) Real-time PCR reaction: prepare reaction system in PCR tube: commercial Taq enzyme PCR Mix (2x) 10 μl, primer mixture 1.6 μl, probe 10.5 μl, template cDNA / negative control reagent / positive control reagent 2 μl, Make up to 20μl with sterilized deionized water;
[0210] The above-mentioned primer mixture contains the above-mentioned 6 specific primers (SEQ ID No.7, SEQ ID ...
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