8.6 Recombinant DNA technology Flashcards
(12 cards)
What is recombinant DNA technology?
Transfer of DNA fragments from one organism to another
Why can transferred DNA be translated within cells of recipient organisms?
• Genetic code is universal
• Transcription and translation mechanisms are universal
How can DNA fragments be produced using restriction enzymes?
• Restriction enzymes cut DNA at specific complementary base recognition sequences either side of target gene, leaving sticky ends
How can DNA fragments be produced from mRNA?
• Isolate mRNA coded for by target gene (easily extracted as more mRNA than DNA and can be transcribed and translated in prokaryotes as introns removed by splicing)
• Mix with DNA nucleotides and reverse transcriptase
• Reverse transcriptase uses mRNA as template to synthesise a single strand of cDNA
• DNA polymerase can form a second strand of DNA using cDNA as template
How can DNA fragments be produced using a gene machine?
• Synthesis fragments of DNA quickly and accurately from scratch
• Amino acid sequence of protein determined, allowing base sequence to be determined
How can DNA fragments be amplified in vitro?
• Polymerase chain reaction (PCR)
• Set up reaction mixture containing DNA fragment, DNA polymerase, primers and DNA nucleotides
• Heat to 95°C to break hydrogen bonds between bases, separating DNA strands
• Cool to 55°C to allow primers to bind to DNA fragment template strand, forming hydrogen bonds between complementary bases
• Heat to 75°C so RNA polymerase joins adjacent nucleotides, forming phosphodiester bonds
• Repeat, doubling amount of DNA each cycle causing an exponential increase
What are primers?
• Short single-stranded DNA fragments
• Complementary to specific DNA base sequences at edge of target gene
• Allowing DNA polymerase to bind
How can DNA fragments be amplified in vivo?
• Add promoter and terminator regions to DNA fragments
• Cut plasmid using same restriction enzymes, leaving sticky ends that can join by complementary base pairing
• Ligase joins DNA fragment to plasmid, forming phosphodiester bonds between adjacent nucleotides
• Insert plasmid into host cell
• Detect transformed / transgenic cells using marker gene
• Culture transformed cells, allowing them to divide and form clones
What common marker genes are used?
• Gene coding for antibiotic resistance as these cells survive antibiotic exposure
• Gene coding for fluorescent proteins as these cells fluoresce under UV light
What are some issues associated with recombinant DNA technology?
• Potential effects on food webs, reducing biodiversity
• Recombinant DNA transferred to weeds
What is gene therapy?
• Introduction of new DNA into cells, often containing healthy alleles
• To overcome effect of faulty alleles in people with genetic disorders
What are some issues associated with gene therapy?
• Effect is short lived as modified cells have limited lifespan
• Immune response against GM cells
• Long term effects not known / negative side effects
