VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip
A detection solution and specific technology, applied in the field of molecular biology, can solve problems such as inability to meet practical applications and cannot be used, and achieve the effects of improving detection accuracy, consistent detection effect, and low cross-reaction rate.
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Embodiment 1
[0020] Embodiment 1 VHL gene mutation detection liquid chip mainly includes:
[0021] 1. ASPE Primers
[0022] Specific primer sequences were designed for wild type and mutant types of six common genotypes of VHL gene T240A, T254C, T266A, C286T, G388C and 444delT. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0023] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1 VHL gene
[0024]
[0025]
[0026] 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.
[0027] 2. Microspheres coated with anti-t...
Embodiment 2
[0038] Example 2 Using the VHL gene mutation detection liquid chip described in Example 1 to detect samples
[0039] The formula of described various solutions is as follows:
[0040] 50mM MES buffer (pH5.0) formula (250ml):
[0041]
[0042] 2×Tm hybridization buffer
[0043] Reagent
source
Final concentration
Dosage per 250ml
1M Tris-HCl, pH8.0
SigmaT3038
0.2M
50ml
5MNaCl
Sigma S5150
0.4M
20ml
Triton X-100
Sigma T8787
0.16%
0.4ml
[0044] Store at 4°C after filtration.
[0045] ExoSAP-IT kit was purchased from US USB Company.
[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0047] 1. Sample DNA extraction:
[0048] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0049] 2. PCR amplification of samples to be tested
[0050] Design 2 pairs of p...
Embodiment 3
[0099] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of VHL gene mutation site
[0100] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)
[0101] Taking the VHL gene T240A and T254C 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 T240A and T254C, and the tag sequence of the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.12, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. 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.
[0102] Table 7 Design of liquid phase chip preparation ...
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