An Alternative Strategy in Rapid DNA Extraction Protocol for High Throughput RAPD Analysis in Chickpea and Its Wild Related Species

Abstract

The fi rst step of RAPD fi ngerprinting is the preparation of the target DNA template. The extraction of DNA from plants has been described by numerous authors. Each author described a different method to overcome the problems for isolation of genomic DNA. Some species like Cicer have certain metabolites like polysaccharides and RNA which interfere with DNA isolation and polymerase chain reaction (PCR) amplifi cation. The objective of this project was to develop high-throughput DNA extraction procedure without the need for greenhouse space or growing Cicer plants. Seeds were germinated of paper fi lter in Petri dish, and 4-day old seedling tissue was used to extract DNA by modifi ed high salt CTAB protocol. Approximately 50-150 ng/μ of genomic DNA was isolated from 2 g of leaf tissue. DNA derived using this method was tested electrophoretically and then was examined with a spectrophotometer: A260/280 (Protein contamination). Polymerase chain reaction amplifi cation was performed using different RAPD primers and EcoRI, MseI and HindIII were used for restriction enzyme reaction. The isolated DNA proved amenable to PCR amplifi cation and restriction digestion. This method does not require expensive reagents and modern laboratory equipments and this technique allows one person to extract nearly 200 storage-stable DNA samples daily, while keeping costs at a minimum. This technique is fast, reproducible, and can be applied for RAPD analysis and most of genetic assays

Keywords

• DNA extraction, chickpea, Cicer species, RAPD, restriction enzyme