Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene
A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of high false positive rate, easy sample contamination, low sensitivity, etc., and achieve the effect of good detection specificity, consistent detection effect and simple steps
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Embodiment 1
[0019] Embodiment 1 STK39 gene SNP detection liquid chip mainly includes:
[0020] 1. ASPE Primers
[0021] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes T107C, G232A and A190C of the STK39 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0022] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 STK395 gene
[0023]
[0024] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.
[0025] 2. Microspheres coated with anti-tag sequences
[00...
Embodiment 2
[0036] Example 2 Detection of samples using STK39 gene detection liquid chip
[0037] The formula of described various solutions is as follows:
[0038] 50mM MES buffer (pH5.0) formulation (250mL):
[0039]
[0040] 2×Tm hybridization buffer
[0041] Reagent
[0042] Store at 4°C after filtration.
[0043] ExoSAP-IT kit was purchased from US USB Company.
[0044] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0045] 1. Sample DNA extraction
[0046] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0047] 2. PCR amplification of samples to be tested
[0048] Three pairs of primers were designed, and multiplex PCR was used to amplify three target sequences of three common genotypes T107C, G232A and A190C containing STK39 gene in one step, and the sequence fragment sizes were 467bp, 400bp and 467bp, respectively. The primer sequences (SEQID NO.19-24)...
Embodiment 3
[0107] The liquid phase chip of embodiment 3 different ASPE primers to the detection of STK39 gene SNP site
[0108] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
[0109] Taking the STK39 gene T107C site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of T107C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.6, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.
[0110] Table 7 Design of liquid phase chip preparation
[0111]
[0112] ...
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