Abstract
Publisher Summary This chapter discusses the use of sulfhydryl Sepharose chromatography for the purification of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences complementary to mecurated recombinant DNA. In the method discussed in the chapter, chromatography is done in jacketed columns of at least 5 × 15 mm. A column is washed extensively in TNE before loading a sample. Sulfhydryl Sepharose can accommodate at least 100/μg of mercurated DNA per milliliter of packed beads and possibly more. After hybridization, the sample is diluted with TNE so that the volume is at least 1 mL and the concentration of mercurated DNA is less than 50 μg/mL. The methodology described in the chapter is potentially useful for the purification of any single-strand DNA or RNA sequence for which a pure homologous probe exists. With this method, highly purified single-strand complementary DNA (cDNA) hybridization probes can be prepared for use in the sensitive quantitation of gene sequences by saturation hybridization. The plus and minus strands of genomic DNA restriction fragments containing sequences homologous to a double-stranded mercurated probe can be highly enriched. In addition, messenger RNAs (mRNAs) or nuclear pre-mRNAs homologous to the probe can be purified for further studies on gene expression.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call