Purification, specific fragmentation, and separation of large DNA molecules.

摘要: Publisher Summary This chapter discusses the processes related to purification, specific fragmentation, and separation of large deoxyribonucleic acid (DNA) molecules. The mentions development three new techniques that allow fractionation analysis DNA molecules on a size scale much larger than previously possible. current methods permit routine handling DNAs up 1.5 million base pairs (bp). first technique involves preparation unbroken genomic inside agarose gels. second digestion such in with restriction nucleases produce discrete, fragments. third technique, pulsed-field gel (PFG) electrophoresis, allows ranging from 10,000 bp (10 kb) more (1.5 Mb). describes demonstrates examples their applicability for bacterial genomes unicellular eukaryotic genomes. procedure preparing intact DNA. Ordinary preparative procedures are carried out solution. In typical preparation, walls cells removed by appropriate enzymatic treatment. resulting spheroblasts or protoplasts then broken open destruction cell membranes detergents metal chelator. produces complex mixture DNA, ribonucleic (RNA), proteins. Treatment proteases RNases may remove some unwanted components. Additional proteins chemical extraction solution phenol, is concentrated alcohol precipitation centrifugation. To obtain clean preparations do not have contaminants, which interfere subsequent analytical preparatory procedures, it frequently necessary repeat these steps. It also essential that, during exposed unnecessarily DNases. electrophoretic chromosomal molecules, fragmentation high-molecular-weight applications technology study organisms simple