Construction of a gene library with mung bean nuclease-treated genomic DNA.

Abstract

AbstractMung bean nuclease (MBN) has been used typically for its single-stranded nuclease activity (1). Under defined reaction conditions, however, which include altered solvation and elevated temperature, hypersensitive sites surrounding coding regions become sensitive to nuclease cleavage (2), while sequences within coding regions remain insensitive Fig. 1). Mung bean cleavage of genomic DNA from Plasmodium species results in a solution of gene-containing fragments Fig. 2) (2). A significant proportion of the noncoding regions is reduced to shards, but it is not known what percentage of the genome this includes. The cleavage sites are not simply single-stranded bubbles in the DNA but stable nucleic acid structures that contain specific arrangements of paired and unpaired nucleotides (3). Size of mung bean vs protein coding regions. Data points from mung bean fragments are shown as dotted circles. Data points from published accounts of other laboratories are shown as open circles. The solid line represents the linear equation, y = 1.2x - 338 derived from the data. The shaded line represents y = x, the expected result if the mung bean fragment sizes were the same size as the coding region. Mung bean nuclease treated of DNA yields products that run as unit bands upon agarose electrophoresis. A plasmid, pPbSL7.8 (2) was treated with EcoRI (lane a), mung bean nuclease and EcoRI (lane b) or mung bean nuclease alone (lane c). The plasmid contains a DNA fragment from P. berghei. The fragment is 7.8 kb and contains the external transcribed spacer, the small subunit rRNA, ITS1, 5.8S RNA, ITS2, and 3 kb of the LSU rRNA. KeywordsMung BeanPlasmodium SpeciesCesium ChlorideMung Bean NucleaseSaponin SolutionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.