Optimization of DNA Extraction from a Single Living Ciliate for Stable and Repetitive PCR Amplification

作者：Kim Se Joo; Min Gi Sik^*

来源：Animal Cells and Systems, 2009, 13(3): 351-356.

DOI：10.1080/19768354.2009.9647229

• Ciliophora
• SSU rDNA
• single cell PCR

Ciliates are undoubtedly one of the most diverse protozoans that play a significant role in ecology. However, molecular examination, based on comparing the DNA sequences, has been done on a limited number of the species. Because most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments. In the present study, we evaluated the effectiveness of four commercial DNA extraction procedures that extract high quality genomic DNA from a single ciliate cell. It was discovered that RED Extract-N-Amp (TM) PCR kit is the best method for removing PCR-inhibiting substances and minimizing DNA loss during purification. This method can also amplify more than 25 reactions of PCR. In addition, this technique was applied to single cells of 19 species belonged to 7 orders under 5 classes that isolated from mixed natural populations. Their small subunit ribosomal DNA (SSU rDNA) was successfully amplified. In summary, we developed a simple technique for the high-yield extraction of purified DNA from a single ciliate cell that may be more useful for rare ciliates, such as tiny and uncultivable marine microbes.

• 出版日期2009-9-30