Cycle 10: DNA Technologies

• Polymerase Chain Reaction (PCR) components


  Primers (extend new strand), dNTPs (4 nucleotides), Taq polymerase (adds dNTPs), MgCl2 (Mg as important polymerase cofactor)
• PCR steps


  Denature + separate strands (95ºC)

  , anneal primers (55ºC), taq pol. binds to DNA and starts synthesis (72ºC)
• Reverse Transcription PCR (RT-PCR) steps


  Lyse cells, extract RNA phase, target poly-a tail, reverse transcription, degrade mRNA, PCR-synthesize DNA
• cDNA (complementary DNA)


  Formed by the reverse transcription of mRNA, only has exons, does not exist in human cells
• RNase (has 2 main functions)


  DNA polymerase activity (requires oligo-DT primer to start), RNase activity (degrades the mRNA from the RNA/DNA hybrids)
• gDNA (genomic DNA)


  Has both introns and exons, exists in the nucleus, obtained using polymerase
• Short Tandem Repeats (STRs)


  Highly polymorphic (unique), short sequences of DNA with variable repetition, children inherit # of STRs from parents (one allele from each parent at a single STR locus)
• Using DNA profiling for sex determination


  Use AMEL: shorter in X, longer in Y
• Making human insulin


  Insert human insulin gene into plasmid (take insulin mRNA, use PT-PCR to generate cDNA), transformed bacteria will produce insulin, insulin is isolated and purified using antibodies
• CRISPR locus


  Has spaces between short palindromic repeats
• Cas9 protein is isolated from

  Bacteria (Streptococcus pyogenes)
• Non-homologous End Joining (NHEJ)


  For a double stranded break, error prone
• Homology Directed Repair


  Donor DNA from lab - can correct a mutation or insert a whole gene into break
• Catalytically impaired Cas9


  Manipulated to only induce a single strand cut since many diseases are caused by a single base mutation
• Goal = CG to TA


  Cytosine deaminase changes C to U, UGI prevents the cell from changing U back to C, now we have UG, nic to remove G and add UA, through DNA replication or repair UA changes to TA
• Application of CRISPR


  Inside the body (ocular disease), outside the body (cancer, create designer t-cells, blood disease)
• Normal t-cells contain PD-1


  Protects tissue from autoimmune attack and facilitate tumour progression
• Steps of PD-1


  Take cells and knock out PD-1 with CRISPR, then reintroduce into the body and the t-cells can now detect and attach the cancer cells