Biomaterial Database

A rapid and efficient method for targeted random mutagenesis. 1988

H Shiraishi, and Y Shimura

Department of Biophysics, Faculty of Science, Kyoto University, Japan.

We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA. A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid. The plasmid is linearized with two adjacent cuts (generating 5' and 3' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region. A non-complementary alpha-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III. Since the nucleotide analogue is resistant to the 3'-5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible. Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized. This procedure was used to mutagenize a specific region of the rnpB gene of E. coli. By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene. The procedure is simple and is applicable to any DNA molecule.

UI	MeSH Term	Description	Entries
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA