Formation of MboII vectors and cassettes using asymmetric MboII linkers.
Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.
Gayle
R B
RB
Department of Biochemistry, Rice University, Houston, TX 77251.
Auger
E A
EA
Gough
G R
GR
Gilham
P T
PT
Bennett
G N
GN
D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008780:Methyltransferases / Q000378:metabolism*; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013379:Substrate Specificity
C047058:DNA modification methylase MboII; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C024413:endodeoxyribonuclease MboII; D015252:Deoxyribonucleases, Type II Site-Specific