Encapsulation of cells in agarose beads for use in the construction of human DNA libraries as yeast artificial chromosomes (YAC).
A simple and general method for the molecular cloning of fragments of over one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was developed for the construction of a human genomic DNA library in a yeast artificial YAC chromosome vector (in situ YAC construction). The main advantages of this method for use in the construction of a human genome library are as follows. First, linear DNA molecules of up to several hundred kilobase pairs in size can easily be prepared by the partial restriction enzyme digestion of the DNA encapsulated in agarose beads in vitro. Second, less than 2 x 10(6) cells scraped from tissue culture plates are sufficient for preparation of the linear DNA molecule for construction of the genome library. The technical manipulations involved in construction of clones of very large segments of DNA, including encapsulation of cells in agarose beads, restriction enzyme digestion, ligation with the YAC vector, transformation into host yeast cells, and stable propagation are discussed.
Yokoyama
K
K
Gene Bank, RIKEN, The Institute of Physical and Chemical Research, Ibaraki, Japan.
Saka
F
F
Kai
T
T
Soeda
E
E
D015139:Blotting, Southern; D002460:Cell Line; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D008863:Microspheres; D012685:Sepharose; D013104:Spheroplasts / Q000235:genetics; D014170:Transformation, Genetic
D004247:DNA; D012685:Sepharose; D004262:DNA Restriction Enzymes