Rapid and inexpensive NaOH based direct PCR for amplification of nuclear and organelle DNA from ramie (Boehmeria nivea), a bast fibre crop containing complex polysaccharides.

摘要

Most of the PCR based approaches in plant science rely on lengthy and expensive DNA isolation protocols, which often involve use of hazardous chemicals. Direct PCR methods save time and cost of experiments and also increase efficiency of PCR. We have compared three rapid DNA extraction processes for direct PCR in ramie (Boehmeria nivea), a fibre crop species containing high amount of gummy complex polysaccharides and developed modified protocols for direct PCR using NaOH as extraction buffer and Tris/Tris-HCl/Tris-EDTA as dilution buffer. These protocols were successful in amplification of nuclear DNA from leaf and stem tissues using ISSR and SSR markers and also chloroplast DNA amplification using primers for rbcL gene. Our results also show that nature and quantity of dilution buffer are important for increasing efficiency of direct PCR. The NaOH based methods are simpler, cheaper and economical compared to other direct PCR methods and work very well for tissues containing high amount of complex polysaccharides. The protocols are suitable for batch processing and high throughput genotyping within a short time period, which will have many applications in plant genomic researches.