6.3.6 genetic engineering Flashcards
define DNA ligase
enzyme that catalyses joining of sugar & phosphate groups within DNA
define electroporation
method for introducing vector with novel gene into cell
- pulse of electricity makes recipient cell membrane more porous
define plasmids
small loops of DNA in prokaryotic cells
define recombinant DNA
composite DNA molecule created in vitro by joining foreign DNA with vector molecule (eg. plasmid)
define restriction enzymes
endonuclease enzymes that cleave DNA molecules at specific recognition sites
define vector
in gene technology, anything that can carry/insert DNA into host organism (eg. plasmids, viruses, certain bacteria)
4 principles of genetic engineering (recombinant DNA technology/genetic modification)
- required gene is obtained
- copy of gene placed inside vector
- vector carries gene into recipient cell
- recipient expresses novel gene
describe step 1 of genetic engineering: obtaining required gene
◦ mRNA obtained from cells where genes being expressed & enzyme (reverse transcriptase) catalyses formation of single strand of complementary DNA (cDNA) using mRNA as template
‣ addition of primers & RNA polymerase makes cDNA into double-stranded length of DNA, whose base sequence
codes for original protein
◦ if know nucleotide sequence of gene, the gene can be synthesised using automated polynucleotide synthesiser
◦ if know sequence of gene, can design polymerase chain reaction (PCR) primers to amplify gene from genomic DNA
◦ DNA probe can be used to locate gene within genome & gene can then be cut out using restriction enzymes
describe step 2 of genetic engineering: placing gene into vector
◦ plasmids obtained from organisms (eg. bacteria) & mixed with restriction enzymes that cut plasmid at specific recognition sites
◦ cut plasmid has exposed unpaired nucleotide bases = sticky ends
◦ if free nucleotide bases, complementary to sticky ends of plasmid, are added to ends of gene to be inserted, the gene & cut plasmid should anneal (bind)
‣ DNA ligase enzymes catalyse annealing
◦ gene may be sealed into attenuated virus which could carry it to host cell
describe step 3 of genetic engineering: getting vector into recipient cell
DNA doesn’t easily recipient cell’s plasma membrane, so various methods used:
◦ heat shock treatment = if bacteria subjected to alternating periods of cold (0 degrees) & heat (42 degrees) in
presence of calcium chloride, their walls & membranes become more porous, allowing in recombinant vector
◦ electroporation = high voltage pulse applied to cell to disrupt membrane
◦ electrofusion = electrical fields help introduce DNA into cells
◦ transfection = DNA can be packaged into bacteriophage, which can then transfect host cell
◦ T1 (recombinant) plasmids are inserted into bacterium Agrobacterium tumefaciens, which infects some plants &
naturaly inserts genome into host cell genomes
describe step 4 of genetic engineering: direct method of introducing gene into recipient
◦ if plants not susceptible to A. tumefaciens, direct methods can be used
◦ small pieces of gold or tungsten are coated with DNA & shot into plant cells = ‘gene gun’
describe reverse transcriptase
• retroviruses (eg. HIV) which contain RNA that they inject into host genome, have reverse transcriptase enzymes which catalyses production of cDNA using their RNA as a template
• reverse of transcription
• enzymes are useful for genetic engineering
describe restriction enzymes
• bacteria & archaea have restriction enzymes (= restriction endonucleases) to protect them from attack by phage viruses
• cut up foreign viral DNA, by process called restriction, preventing viruses making copies
• prokaryotic DNA protected from action of these enzymes by being methylated at recognition sites
• useful for molecular biology & biotechnology as molecular scissors, as they reconnect specific sequences within length of DNA & cleave molecule there
• some make staggered cut (sticky end) & others produce blunt ends
• always recognise palindromic sequence; reading 2 strands of DNA in same orientation (eg. 5’ to 3’)
• some require magnesium as a cofactor
describe ligase enzymes
• used in molecular biology to join DNA fragments
◦ catalyses codensation reactions which join sugar groups & phosphate groups of DNA backbone
• catalyse such reactions during DNA replication in cells & also used in PCR
how can insulin be produced from GM bacteria
obtain mRNA from beta cells of islets of Langerhans in human pancreas (where insulin is made):
- adding reverse transcriptase enzyme to mRNA makes single strand of cDNA & treatment with DNA polymerase makes a double strand (the gene)
- addition of free unpaired nucleotides at ends of DNA produces sticky ends
- with hep of ligase enzymes, the insulin gene can be inserted into plasmids extracted from E. coli bacteria
◦ called recombinant plasmids (contain inserted DNA) - E. coli bacteria are mixed with recombinant plasmids & subjected to heat shock in presence of calcium
chloride ions, so they take up the plasmids
= genetically modified bacteria cultured in large numbers to produce insulin
