Primer, kit and method for detecting APC gene mutation
A kit and reagent technology, applied in the field of primers and kits for detecting APC gene mutation, can solve the problems of tediousness, inability to do prenatal diagnosis, time-consuming, etc. Effect
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Embodiment 1
[0078] A test kit for detecting APC gene mutation in familial adenomatous polyposis, comprising the following components:
[0079] (1) Perform PCR amplification on the 16 exon coding regions of the APC gene. Since the No. 16 exon is relatively large, 31 pairs of primers (nucleotide sequences shown in sequence as SEQ ID NO.1-62) were designed for Amplify.
[0080] (2) PCR amplification reagents: 10×PCR bμffer, dNTP, Takara LA Taq enzyme;
[0081] (3) PCR product purification reagent: ordinary agarose gel DNA recovery kit (spin column type) (Tiangen, DP209);
[0082] (4) Sequencing reagents: 2.2 μL BigDyemix (Bigdye, 5×seq), 2.5 μL EDTA, 30 μL ethanol solution (100%), 100 μL ethanol solution (70%), 8 μL Hi-Di.
[0083] This kit is stored at -20°C, and repeated freezing and thawing should be avoided as much as possible.
[0084] All of the above primer pairs can amplify the APC gene sequence shown in NCBI Reference Sequence: NM_000038.5.
Embodiment 2
[0086] A test kit for detecting the APC gene mutation of familial adenomatous polyposis, the steps for using it include the following steps:
[0087] (1) Sample genomic DNA extraction
[0088] The genomic DNA of the samples was extracted using a blood genomic DNA extraction kit (0.1-1 ml) (Tiangen, DP318).
[0089] (2) Different primer pairs are used for PCR amplification of APC gene
[0090] Primers (as shown in SEQ ID NO.1-62) were designed for the APC gene for PCR amplification. The 15 μL PCR reaction system includes: 1.5 μL of 10×PCR bμffer, 2.4 μL of dNTPs, 0.15 μL of Takara LA Taq enzyme, 0.3 μL of upper and lower primers (10 μM), 0.5 μL of genomic DNA (100 ng / μL), and ddH 2 O complements the reaction system.
[0091] The PCR reaction program was: 94°C pre-denaturation for 15 minutes; 94°C denaturation for 30 seconds, 62°C annealing for 30 seconds, 72°C extension for 45 seconds, 14 cycles; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension...
Embodiment 3
[0103] APC gene amplification primer screening experiment, including the following primer sequences and reagents:
[0104] (1) There are 8 pairs of primers for the coding region of exons 5, 8 and 16 of the APC gene, and the nucleotide sequences thereof are shown in SEQ ID NO.63-78.
[0105] (2) PCR amplification reagents: 10×PCR bμffer, dNTP, Takara LA Taq enzyme;
[0106] (3) PCR product purification reagent: ordinary agarose gel DNA recovery kit (spin column type) (Tiangen, DP209);
[0107] (4) Sequencing reagents: 2.2 μL BigDyemix (Bigdye, 5×seq), 2.5 μL EDTA, 30 μL ethanol solution (100%), 100 μL ethanol solution (70%), 8 μL Hi-Di.
[0108] APC gene amplification primer screening experimental steps include:
[0109] (1) Sample genomic DNA extraction, same as in Example 2.
[0110] (2) APC gene PCR amplification, the same as in Example 2.
[0111] (3) Purification of the PCR product is the same as in Example 2.
[0112] (4) DNA sequencing reaction, same as in Example 2...
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