Gene changes can help identify which genes are performing which functions.
process
Plasmids and other vectors can have genes. Added chemicals or enzymes can mutate genes {in vitro mutagenesis}. Vectors go into hosts, express genes, and make protein.
methods
Gene changes can be at restriction endonuclease sites. If sites have overhanging strands, S1 nuclease can remove overhanging single-strand DNA, or DNA polymerase can extend shorter strands, to make blunt ends. Linkers can attach to blunt ends.
Chemicals can alter gene nucleotides. Sodium bisulfite makes C into U. Hydrazine and formic acid delete nucleotide nitrogenous base, leaving sugar and phosphate. At low nucleotide concentrations or in harsh chemical conditions, DNA polymerase can add wrong nucleotides during DNA synthesis.
In vitro mutagenesis {site-directed mutagenesis} can study binding sites and functional regions. Site-directed mutagenesis hybridizes synthetic 10-base to 15-base oligonucleotides to DNA sites. Oligonucleotides differ from original sequences by one nucleotide at end. Oligonucleotides hybridize well to original sequences, because they differ by only one nucleotide. Hybridized sequences replicate to make mutated genes.
enzymes
DNA ligase connects perfectly aligned DNA strands. Mutated ends do not ligate {ligase-mediated}, showing mutation locations. RNase A cuts DNA-RNA complexes where sequences mismatch and can detect mutation locations. Osmium tetroxide and hydroxlamine cut at unmatched C or T bases. Restriction enzymes fragment single-strand DNA. Different DNA fragments have different conformations and so different mobilities {single-stranded conformation polymorphism} (SSCP).
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Date Modified: 2022.0225