LEAP: L1 Element Amplification Protocol.
LEAP: L1 Element Amplification Protocol.
复制标题
DOI：
10.1007/978-1-4939-3372-3_21
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Moran, John V
中科院分区：
文献类型：
--
作者：
Kopera, Huira C;Flasch, Diane A;Nakamura, Mitsuhiro;Miyoshi, Tomoichiro;Doucet, Aurelien J;Moran, John V
长散布元件-1（LINE-1或L1）反转录转座子编码反转录转座所需的两种蛋白质（ORF 1 p和ORF 2 p）。L1元件扩增方案（LEAP）测定L1 ORF 2 p在体外逆转录L1 RNA的能力。超离心或免疫沉淀用于从用工程化的L1表达构建体转染的培养的人细胞中分离L1核糖核蛋白颗粒（RNP）复合物。将分离的RNP与在其5′端含有独特序列且在其3′端含有富含胸苷序列的寡核苷酸一起孵育。向反应物中加入dNTP使得L1 ORF 2 p与L1 RNA结合以产生L1 cDNA。然后使用聚合酶链式反应（PCR）扩增所得的L1 cDNA，并通过凝胶电泳观察产物。然后对所得PCR产物进行测序，以进行产物验证。LEAP检测有助于确定L1 ORF 1 p和ORF 2 p突变如何影响L1逆转录酶（RT）活性。此外，LEAP分析表明，L1 ORF 2 p RT在与L1 RNA模板退火时可以延伸具有错配的3′端碱基的DNA引物。由于LINE-1生物学领域倾向于研究调节LINE-1的细胞蛋白，分子遗传学和生物化学方法，如LEAP，结合LINE-1培养细胞逆转录转座试验，对于剖析L1逆转录转座的分子机制至关重要。
Long Interspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 Element Amplification Protocol (LEAP) assays the ability of L1 ORF2p to reverse transcribe L1 RNA in vitro. Ultracentrifugation or immunoprecipitation is used to isolate L1 ribonucleoprotein particle (RNP) complexes from cultured human cells transfected with an engineered L1 expression construct. The isolated RNPs are incubated with an oligonucleotide that contains a unique sequence at its 5′ end and a thymidine-rich sequence at its 3′ end. The addition of dNTPs to the reaction allows L1 ORF2p bound to L1 RNA to generate L1 cDNA. The resultant L1 cDNAs then are amplified using polymerase chain reaction (PCR) and the products are visualized by gel electrophoresis. Sequencing the resultant PCR products then allows product verification. The LEAP assay has been instrumental in determining how mutations in L1 ORF1p and ORF2p affect L1 reverse transcriptase (RT) activity. Furthermore, the LEAP assay has revealed that the L1 ORF2p RT can extend a DNA primer with mismatched 3′ terminal bases when it is annealed to an L1 RNA template. As the LINE-1 biology field gravitates toward studying cellular proteins that regulate LINE-1, molecular genetic and biochemical approaches such as LEAP, in conjunction with the LINE-1 cultured cell retrotransposition assay, are essential to dissect the molecular mechanism of L1 retrotransposition.