Primer composition for detecting harmful gene of deficiency of uridine monophosphate synthase of cattle, kit with primer composition and application of kit
A technology of bovine uridylate synthase and primer combination, which is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, can solve the effect of PCR amplification efficiency, prone to error fragments, amplification Problems such as low efficiency, to achieve the effect of excellent amplification specificity, ensuring accuracy, and high amplification efficiency
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Embodiment 1
[0034] Example 1 Preparation of nested PCR reaction kit for screening tauridylate synthase deficiency
[0035] 1. Primer design
[0036] According to the UMPS gene sequence on NCBI (GenBank: AC_000158) combined with the overall idea of the present invention, continuously analyze, summarize and adjust the design of primers, and finally according to the C-terminal code of uridine synthase (UMPS) gene on bovine chromosome 1 (BTA1) There is a C→T point mutation at sub-405, and a nested PCR method for detecting the harmful gene of uridine synthase deficiency was designed, and primer 1, primer 2, primer 3 and primer 4 were obtained. The sequences are respectively shown as SEQ ID NO.1-4:
[0037] Primer 1, Primer 2, Primer 3 and Primer 4 were designed according to the bovine gene sequence on NCBI. The sequence is shown as SEQ ID NO.1~4:
[0038] Primer 1: SEQ ID NO.1: gttattttag ggtcttagtg gagc;
[0039] Primer 2: SEQ ID NO.2: atatttcaat aaaaagtaac c;
[0040] Primer 3: SEQ ID...
Embodiment 2
[0054] Example 2 Using the nested PCR reaction kit prepared in Example 1 to detect cow uridine synthase deficiency
[0055] 1. Extraction of whole DNA from bovine blood
[0056] The jugular vein blood sampling method randomly collected blood samples of 243 Holstein cows in an isolation farm in Jiangsu, 5mL / head, and placed them in vacuum heparin sodium anticoagulant tubes, shaken well, then divided them into 2mL tubes with centrifuge tubes, and stored them frozen at -20°C for later use .
[0057] Promega Maxwell 16 Automatic Nucleic Acid Extractor was used to extract blood genomic DNA, and a matching kit (AS1010 from Promega Company) was used. Promega's AS1010 kit contains Maxwell 16 Blood DNA Cartridges, Purification Plungers, Elution Tubes, and Elution buffer. The operation was carried out according to the instructions of the kit, and the DNA extraction of 16 samples could be completed approximately every 28 minutes after the instrument was started. After the DNA is ext...
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