General multiplex polymerase chain reaction realization method based on microarray chip
A microarray chip and chain reaction technology, applied in the field of biomedicine, can solve the problems that the instrument cannot be applied on a large scale, the maximum multiplicity is low, and the operation is complicated.
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[0033] Example 1: Simultaneous amplification of 9 exons of Duchenne muscular dystrophy (DMD) gene
[0034] The sample is the blood of healthy people, and the genomic DNA is extracted using the rapid DNA extraction and amplification kit of Tiangen Biochemical Technology Co., Ltd. This embodiment includes the following steps:
[0035] The first step is to use the microarray chip to spot multiple pairs of primers into several hydrophilic micropores respectively, and then add the PCR components to the hydrophilic micropores by pulling or drainage;
[0036] The microarray chip spot sample comes from Boao Biological Co., Ltd., the model is SmartArrayer TM 48.
[0037] Such as figure 2 As shown, the microarray chip includes a substrate, a hydrophobic surface and micropores, wherein the hydrophobic surface is located on the top of the substrate, and a plurality of micropores are arranged in an array throughout the hydrophobic surface.
[0038] The microarray chip is prepared in the followin...
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[0059] Example 2: Multiple amplification and chip detection of cloned plasmids of 11 pneumonia pathogens
[0060] 1. Sample:
[0061] The cloned plasmids of 11 pneumonia pathogens (see the table below) corresponded as templates. The vector is pGM-T, from Tiangen Biochemical Technology Co., Ltd. Plasmid extraction kit (Tiangen Biochemical) was used to extract plasmid.
[0062] Numbering
[0063] Numbering
[0064] 2. Primers and probes
[0065] All primers and probes are synthesized by Shanghai Shenggong and purified by PAGE.
[0066] Universal primers: upstream primer 5'TCACTTGCTTCCGTTGTCC 3', downstream primer 5'GGTTTCGGAT GTTACAGGCA 3'.
[0067] Specific primers: According to the sequence of each target fragment, use the primer design software Primer Premier 5 to design by yourself, the sequence information is as follows:
[0068] Name
[0069] Name
[0070] Specific probes for DNA chips (2 for each template), the sequence is as follows:
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[0072]
[0073] 3. Constr...
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