Poppy species specificity genetic marker detecting system
A poppy, target sequence technology, used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of high accuracy, accurate amplification, high sensitivity and stability
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Embodiment 1
[0053] Embodiment 1, STR composite amplification primer design
[0054] 1. Design principles
[0055] Suitable poppy STR loci were screened according to the following principles:
[0056] (1) The repeating sequence of the locus is simple, and the repeating unit is a locus with two, three, four or five nucleotides;
[0057] The size of the amplified fragment of the gene locus is between 90 and 400 bp. In order to prevent overlapping between the loci, there should be at least 9 to 10 bp interval between the loci of the same fluorescent combination;
[0058] (2) The locus mutation rate is low and the polymorphism is good, ensuring the stability and reliability of the test results;
[0059] (3) The primers of each locus must be compatible, that is, the primers do not interact, especially the complementary sequences cannot appear at the 3' end of the primers; because the special sequences between the primers of the loci interact, such as primer dimers and hairpin structures. Som...
Embodiment 2
[0068] Embodiment 2, the application of STR composite amplification primers in the specific identification of poppy species
[0069] 1. Genomic DNA extraction
[0070] Genomic DNA was extracted separately from the flowers, stems, leaves and seeds of the poppy samples.
[0071] 2. Optimization of STR multiplex amplification system
[0072] Using the genomic DNA of poppy leaves as a template, the primer pairs P5, P12, P13, and P14 were mixed for PCR amplification, and the amplification system and reaction conditions were optimized, as follows:
[0073] The amplification system before optimization is shown in Table 2:
[0074] Table 2 Amplification system before optimization
[0075]
[0076] The STR multiplex amplification reaction conditions before optimization are as follows:
[0077] Initial denaturation conditions: 92-96°C, 5-11 minutes;
[0078] Denaturation conditions: 91-97°C, 30 seconds-2 minutes;
[0079] Annealing conditions: 53-62°C, 30 seconds-2 minutes;
...
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