| 摘要: |
| 以天蓝喇叭虫(Stentor coeruleus)为研究对象,探索了单细胞单基因PCR扩增及单细胞全基因组PCR扩增技术在原生动物中的应用.经过不断探索和优化条件后,试验取得了理想的结果.在40例单细胞单基因(SSU rDNA基因全序列)PCR的一次性扩增中,新鲜细胞和经过中性红染色的细胞都获得了100%的成功率,室温下酒精(95%)保存一周的细胞获得了82.5%的成功率.在单细胞全基因组PCR扩增中,采用高效高保真的phi29DNA聚合酶结合随机引物(Random Primer)进行扩增,获得了丰富且质量较高的PCR产物.以全基因组扩增产物(稀释10倍)对四个常用的基因位点(TEF1、SSU rRNA、18S-ITS1-5.8S、a-tubulin)进行扩增,均成功获得相应的基因片断. |
| 关键词: 单细胞PCR 单细胞全基因组扩增 原生动物 天蓝喇叭虫 |
| DOI：10.18307/2008.0517 |
| 分类号: |
| 基金项目:国家自然科学基金项目(30490232)资助 |
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| A preliminary study of single cell PCR in protozoa: take Stentor coeruleus for an instance |
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LIU Zhi-xin1,2, YU Yu-he1
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1.Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P.R.China;2.Graduate School of Chinese Academy of Sciences, Beijing 100049, P.R.China
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| Abstract: |
| Taking Stentor coeruleus for an instance, we have tested single cell PCR for single gene and whole genome. After optimization, we got perfect results. In the single cell PCR for single gene reaction, 40 fresh cells and 40 neutral red stained cells all got 100% success and rich products, and 40 cells kept in the alcohol (95%) for one week got 82.5% success. In the single cell PCR for whole genome reaction, we employed phi 29 DNA Polymerase and random primer for our PCR amplification and got rich products. Using whole genome amplification (10 times diluted) 4 gene loci were amplified, and all of them got objective gene section successfully. |
| Key words: Single cell PCR whole genome amplification Protozoa Stentor coeruleus |
