DNA萃取方式以及分離葉綠體DNA方法在非光合作用植物中的比較與應用

Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

利用三種植物DNA抽取套組及三種以CTAB為基礎的萃取方式，萃取兩種非光合作用植物日本蛇菰（Balanophora japonica）和菱形奴草（Mitrastemon kanehirai）的DNA，隨後測試並比較各種不同方式抽取所得DNA的品質。所有方法抽取出的DNA品質均足以進行PCR；而利用Barnwell等人（1998）針對富含黏液的多肉植物發展出的方法所抽出的蛇菰及奴草DNA，其純度皆能夠進行限制酶切割反應。此外我們採取了Milligan的方法成功地抽取出富含質體DNA（plastid DNA）的菱形奴草DNA。這些基於Milligan在1989年所敘述利用高鹽溶液以去除細胞核DNA的方法能夠有效的增加質體DNA，並且明顯減少了細胞核DNA的含量。這種增加質體DNA的萃取方式不但花費低廉且不需耗費大量時間，並有相當大的可能性能夠成功的運用在其他非光合作用植物上。

Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998) performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989) were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.