8.6 Recombinant DNA technology Flashcards
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
