A general method to cleave a known DNA sequence at any site.
We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector.
Freije
J M
JM
Departamento de Biología Funcional, Facultad de Medicina Universidad de Oviedo, Spain.
Muñoz
M
M
López-Otín
C
C
Viñuela
E
E
D000358:African Swine Fever Virus / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003854:Deoxyribonucleotides; D004815:Epidermal Growth Factor / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D010957:Plasmids
D003854:Deoxyribonucleotides; D004815:Epidermal Growth Factor; D004247:DNA; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes