Isolation, purification and characterization of DNA from the Capsicum species found in the Philippines

• Main Authors: Calpe, Ma. Michelle Anne A., Chow, Jeanne Valerie Clichy Y.
• Format: text
• Language: English
• Published: Animo Repository 2004
• Subjects: Peppers > Philippines
• Online Access: https://animorepository.dlsu.edu.ph/etd_bachelors/9536
• Institution: De La Salle University

Isolating high-quality genomic DNA from Capsicum species was found to be difficult. Common DNA extraction procedures have produced poor yields because of dehydrated leaves accumulate polysaccharide and RNAses. This study has proposed an optimized condition for the isolation procedure yielding to a relatively pure DNA from Bell pepper and capsicum annum's leaves. The presence of DNA was then verified by means of measuring its absorbance at 260 nm using UV-Vis spectrometer. The isolate DNA was then subjected to restriction digestion before characterizing its genetic profile by determining its molecular weight. Denaturing High-Performance Liquid Chromatography and agarose gel electrophoresis are the techniques used for analyzing the DNA fragments. It appeared that the use of EcoRI and HindIII as restriction endonucleases were able to digest the isolated DNA. DHPLC was not an effective technique for analyzing large genomic DNA. It was found to be applicable only to chromosomal DNA. Agarose gel electrophoresis is a technique used for the separation and visualization of DNA bands. The utilization of Blue view nucleic acid resulted with no visible bands. However, ethidium bromide, which is an intercalating agent, was found to resolve the desired bands. The DNA bands appeared after the staining process and were viewed using UV-Vis transilluminator. At 23130 bp, a distinct band was seen which would most likely be the most important for the Capsicum species. The activity of RNAse was also determined. it showed no significant difference in the results of the samples with and without the said enzyme and that it has no inhibitory effect in the restriction enzymes used in digestion. Documentation of the gel made use of a digital camera. Pulse-Field electrophoresis is suggested to attain better band migration and separation for further studying and eventually establishing a genetic marker for the Capsicum species.