Partial Cvi JI Digestion as an Alternative Approach to Generate Cosmid Sublibraries for Large-Scale Sequencing Projects

Abstract

We demonstrate an alternative method for the generation of random subclones for large-scale shotgun human DNA sequencing projects. Miniprep DNA from a human cosmid clone was partially digested with CviJI, size-fractionated by agarose gel electrophoresis and cloned into bacteriophage M13. A library consisting of 10(5) subclones of 1.1 kb average size was recovered from one gel fraction containing approximately 300 ng of partially digested DNA. DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the subclones were generated at random. DNA sequences from three of the subclones did not match the cosmid sequences, establishing that miniprep DNA can be used for library construction with little concern for contamination with genomic E. coli DNA. The use of CviJI to generate random DNA fragments thus offers a simple alternative to other commonly used fragmentation methods, such as shearing or sonication, for the generation of random sublibraries for large-scale human shotgun DNA sequencing projects.

A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries

“…One explanation for this behavior, common to all these enzymes, might be steric limitations imposed by cleavage of the very short DNA substrates dominating the reaction during the course of digestion [36]. Even under partial digestion conditions, however, very frequent cutters are still useful for genomic library preparation, yielding a quasi-random accumulation of DNA sequences ( [20,21,37]; this work) they are also of value in other cloning technologies, including ultrasensitive DNA; labeling/amplification [20], highest resolution restriction mapping [19,20], RFLP, single-copy gene amplification, detection/identification of non-cultured pathogenic microorganisms [19,22] and for increasing the limited pool of commercially available Type II REases specificities.…”

Chloroviruses

“…One of the restriction endonucleases, CviJI, is interesting because under certain conditions (referred to as CviJI*) it cleaves PuGCPy, PyGCPy, and PuGCPu sequences between the G and C, thus producing blunt ended DNA fragments. Consequently, CviJI* has been used for shotgun cloning of DNA because it requires less DNA and avoids DNA end repair processes [86][87][88]). Two of the virus-encoded DNA endonucleases are unusual because they cleave one strand of the duplex DNA in a site-specific manner, i.e., they are nicking enzymes, Nt.CviPII (/CCD) [89] and Nt.CviQXI (R/AG) [81].…”

Evaluation of single-nucleotide polymorphisms in CAPN1 for association with meat tenderness in cattle1,2

“…A random shotgun library was prepared from a Bacterial Artificial Chromosome (BAC) clone (Smith et al, 2000a;Warren et al, 2000) containing the CAPN1 gene by partial digestion of~5 g of BAC DNA, with the restriction enzyme CviJI (0.3 units per L) essentially as described (Gingrich et al, 1996). Briefly, the DNA was incubated with enzyme in a 50-L volume for 20 min, the resulting DNA smear was separated on a 1% agarose gel, and fragments in the 1 to1.5 kb range were isolated using a commercial kit (Novagen, Madison, WI).…”