摘要
试验旨在建立一种适合于蒙古绵羊肌内脂肪沉积相关基因mRNA表达量的检测方法。根据GenBank中脂肪沉积相关基因的序列,分别设计1对特异性引物,然后通过克隆测序检测引物扩增片段的正确性,最后使用实时荧光定量PCR(Real-time PCR)技术验证方法的可行性。结果显示,最佳的PCR反应参数为:94℃预变性4 min;94℃变性30 s,58℃退火45 s,72℃延伸1 min,40个循环;72℃延伸5 min。各目的基因的熔解曲线峰值单一,目的产物稳定。结果表明成功建立了一种快速检测蒙古绵羊肌内脂肪沉积基因的Real-time PCR方法,可用于绵羊肌内脂肪沉积基因的检测及定量分析。
This experiment was conducted to establish a detection method which was suitable for genes expression of Mongolia sheep intramuscular fat （IMF） deposition genes. According to the sequences of IMF deposition genes in GenBank, pairs of specific primers were designed and synthesized. Then the amplified fragments were detected by cloning technology to ensure the accuracy of the PCR products. The Real-time fluorescent quantitative PCR （Real-time PCR） was used to check the feasibility of method. Results showed that Real-time PCR optimum cycle parameters were 94 ℃ 4 min, 94 ℃ 30 s, 58 ℃ 45 s, 72 ℃ 1 min,40 cycles;72 ℃ 5 min.Each peak melting curve was a single gene, the target product was stable. The method of Real-time PCR was established successfully and could be specially used to detect IMF deposition genes of Mongolia sheep.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第9期37-41,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金资助项目(30860194)
