This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.
Comparison of different procedures for DNA extraction for routine diagnosis of phytoplasmas
Carmine Marcone
2019-01-01
Abstract
This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
