BackGenetics: Genetic Engineering and Molecular Techniques
Terms in this set (20)
Genetic engineering is the intentional modification of DNA to produce new traits or study gene function by combining DNA from different organisms to form recombinant DNA.
Recombinant DNA is a combined DNA molecule formed by joining DNA fragments from different sources, often using restriction enzymes and ligase.
A vector is a DNA carrier, usually a plasmid, used to transport a DNA fragment (gene of interest) into host cells for cloning or expression.
Important plasmid features include an origin of replication, a multiple cloning site (MCS) with restriction sites, and an antibiotic resistance gene like AmpR for selection.
Restriction enzymes recognize specific DNA sequences and cut DNA, often creating sticky ends that allow complementary DNA fragments to anneal for recombinant DNA formation.
Sticky ends are short single-stranded DNA overhangs created by staggered cuts from restriction enzymes, enabling complementary base pairing between DNA fragments.
DNA ligase seals the sugar-phosphate backbone by forming phosphodiester bonds between DNA fragments after sticky ends anneal, creating stable recombinant DNA.
Transformation is the process where bacteria take up plasmid DNA, allowing propagation of recombinant plasmids and formation of colonies on selective media.
In blue-white selection, bacteria with plasmids containing intact lacZ produce blue colonies; insertion of foreign DNA disrupts lacZ, producing white colonies, both resistant to antibiotics.
Prokaryotes usually lack introns and require minimal RNA processing, allowing direct genomic cloning; eukaryotes have introns and require cDNA (from spliced mRNA) for bacterial expression.
cDNA is complementary DNA synthesized from mature mRNA using reverse transcriptase, poly-dT primers, RNase H, DNA polymerase, and ligase, containing only exons.
Gibson Assembly is a cloning method that joins multiple DNA fragments with overlapping ends using exonuclease, DNA polymerase, and DNA ligase without restriction enzymes.
The Bt gene from Bacillus thuringiensis encodes insecticidal toxins; it is inserted into crops like corn to create pest-resistant genetically modified plants.
Reproductive cloning creates genetically identical organisms, differing from molecular cloning which clones only DNA fragments or genes.
SCNT involves removing the nucleus from an egg cell and inserting a somatic cell nucleus, allowing the embryo to develop into a clone of the donor organism.
Dolly was the first mammal cloned by SCNT, demonstrating that differentiated somatic cells contain a complete genome capable of directing development.
Extrachromosomal arrays are large DNA arrays formed from injected DNA that behave like chromosomes but are unstable and can cause mosaicism.
MosSCI inserts a single copy of a transgene directly into the genome, providing stable inheritance and normal expression compared to extrachromosomal arrays.
P-elements are transposable elements used to insert genes into the fly genome, requiring transposase and inverted repeats flanking the gene of interest.
Gene targeting uses homologous recombination with targeting vectors containing homologous DNA to insert, replace, or knockout specific genes in the genome.