Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.
An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.
LaBanca
F
F
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-2131, USA.
Krishnan
B R
BR
Chaplin
D D
DD
Berg
D E
DE
Berg
C M
CM
D000818:Animals; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements; D005821:Genetic Techniques; D006680:HLA Antigens / Q000235:genetics*; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D008285:Major Histocompatibility Complex; D016296:Mutagenesis; D015183:Restriction Mapping; D017384:Sequence Deletion
D004251:DNA Transposable Elements; D006680:HLA Antigens; D004262:DNA Restriction Enzymes