20 Genetic Engineering Flashcards
genetic engineering
rearrangement of DNA - not writing/creating
- often inter-special - eg. human insulin in bacteria
- works bc all prokaryote and eukaryote share same genetic bases
what 2 biological tools are used to cut + paste DNA
- Restriction endonuclease
- DNA ligases
REases with blunt cutting
no staggered cuts - still palindromic - not as useful
Restriction endonucleases:
- Recognise palindromic sequences - same sequence on complementary stands going opposite directions, between same 2 bases cut on both strands = staggered cuts = overhangs, e.g., G/A, A/G (both left to right)
- Like enzymes each REase recognises a specific sequence
- Naturally produced by bacteria to fight off viral infections (phages) - REase recognises + breaks down viral DNA, but coexpresses an MTase to methylate + protect their own DNA
DNA Ligase
- stitches together 2 bits of DNA together - forms phosphodiester bonds between 5’ phosphate (top right) and 3’ hydroxyl (OH group) (bottom left)
- works better on (overhangs from) staggered cuts from REs bc DNA strands are already connected/held together (can do but is less effective)
why can different restriction enzymes (endonucleases) be used to spice different strands together?
different RE cut at the same codes to create the same overhangs which can ligate together
Vectors
carriers/ hosts to the recombinant DNA to produce recombinant protein e.g., (bacteria) plasmids (easy to transfer from one cell to another)
structure of a plasmid (‘expression vector’)
ring of non essential DNA to its survival, contains:
- origin of replication
- advantageous gene e.g., antibiotic resistance
- promoter region for REs
- restriction enzyme sites (when used for gen. engineering) - contains promoter region + multiple cloning site (sequence that recognises many diff. REs
process of engineering a vector
- cut specified gene out of original strand with RE, use same RE to target vector, use ligase to bond complimentary ends.
Using PCR in cut + paste RE sites from DNA strand to vector
- Use PCR to clone target gene (to be inserted)
- PCR mixutre includes primers with incorporated RE sites to bind on original DNA to each side of the gene, copied in as part of the new strands on either end of gene
- then pasted using RE binding to sites to cut + paste into vector + ligate
what is site directed mutagenesis?
process used to make specific changes (substitutions/insertions/deletions of a few nucleotides) to DNA sequence - not just rearrangement!
- methylated original plasmid, copied to new DNA with added in primers,
- enzymes added to digest away old methylated plasmid
