c-KIT gene mutation detection liquid-phase chip
A detection solution and chip technology, applied in the field of molecular biology, can solve the problems of easy contamination of samples, high false positive rate, high price, etc., and achieve the effects of strong scalability, simple steps, and improved sensitivity.
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Embodiment 1
[0018] The c-KIT gene mutation detection liquid chip described in this embodiment mainly includes:
[0019] 1. ASPE Primers
[0020] Specific primer sequences were designed for the four common mutation sites AY502-503dup, V560G, T670I, and D816H / V of the c-KIT gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0021] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)
[0022]
[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 100pmol / mL stock solution with 10mmol / LTris Buffer.
[0025] 2. Microspheres coated with anti-tag sequences
[0...
Embodiment 2
[0037] Example 2 Detection of samples by c-KIT gene mutation detection liquid chip
[0038] The formula of described various solutions is as follows:
[0039] 50mM MES buffer (pH5.0) formulation (250mL):
[0040]
[0041] 2×Tm hybridization buffer:
[0042]
[0043] Store at 4°C after filtration.
[0044] ExoSAP-IT kit was purchased from US USB Company.
[0045] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0046] 1. Sample DNA extraction:
[0047] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0048] 2. PCR amplification of samples to be tested
[0049] Three pairs of primers were designed using Primer5.0, and the c-KIT genes AY502-503dup, V560G, T670I, and D816H / V were amplified by multiplex PCR in one step. The product sizes were 350bp, 309bp, and 301bp, respectively. The primer sequences (SEQ ID NO. 33) See Table 3 above.
[0050] First prepare m...
Embodiment 3
[0102] The liquid phase chip of embodiment 3 different ASPE primers detects c-KIT gene mutation site
[0103] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
[0104]Taking the c-KIT gene AY502-503dup 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 AY502-503dup, and the Tag sequence at the 5' end of the ASPE primer was selected. From SEQ ID NO.1-SEQ ID NO.9, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.19-SEQ ID NO.27. 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.
[0105] Table 7 Design of liquid phase chip preparation
[0106]
[010...
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