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BIOL 310 - Ch 19 Lecture Notes

  • electrophoresis

    • separates macromolecules based on charge and size

      • DNA is negatively charged and will migrate towards the positive side of the well

      • large molecules migrate slowly, slow molecules migrate fast

    • standard / ladder is used to identify the sizes of the unknown samples

    • uses apparatus, agarose gel, buffer, electricity and stain

    • basic apparatus for DNA

  • restriction enzymes

    • endonucleases — cut DNA at very specific sites

    • type II: palindromic sequences e.g. GAATTC & CTTAAG

      • cohesive or blunt

    • evolved to protect bacteria from foreign DNA

    • EcoRI has recognition site & the EcoRI methylase

  • restriction enzyme uses

    • gene cloning — the isolation and amplification of a given gene in a non-host organism

    • usually in a plasmid

      • small circular DNA

      • referred to as a vector — vector has a selector marker (if the cloning was successful or not); multiple unique RE sites

  • cloning process

    • necessary components: source DNA, acceptor plasmid, bacterial host, and restriction enzymes

    • steps:

      1) restrict gene of interest

      2) restrict vector

      3) mix vector & gene & ligase

      4) introduce recombinant (plasmid DNA & source DNA present) molecule into host

      5) grow host

      6) determine success

  • cloning uses

    • research

    • recombinant DNA molecule

      • in vitro gene expression

      • expression vector is used: putting a gene into a plasmid under promoter and operator system so the bacteria will make that gene (@15:00)

  • cloning vectors

    • plasmids: accept 0.1 to 5kb of DNA insert

    • cosmids: accept 30-40 kb DNA insert

    • BACs: accept 150-300 kb DNA

    • YACs: accept 300-500 kb DNA

      • genomic library (x3 of genes stored to ensure all the DNA is present - some redundancy)

  • polymerase chain reaction (PCR)

    • DNA replication in vitro

    • billions of copies from single template

    • necessary components: template, primers (3’-OH), Taq DNA polymerase (thermophile), free dNTPs, buffer, magnesium, thermal cycler

    • two finite strands (before 25:00)

    • PCR process

      • initial denaturation (94o C for 2 minutes)

      • 30 cycles

        • denaturation (94o C for 30 seconds)

        • primer annealing (50-60o for 30-60 seconds)

        • elongation (68-72o for 1 min/kbp)

      • exponential amplification of DNA

      • copy # = 2n where n = # of cycles

      • cycle vs copies

        • 1= 21= 2

        • 3= 23 = 8

    • DNA denatured, the primers will bind based on polarity for cycle 1; a strand of DNA is created of unknown size (29:00)

      • when amplifying one part of a strand, the next replication cycle already has the ____👎👎

  • applications of PCR

    • diagnosis of inherited human diseases

    • pathogen subtypes

    • identification of individuals in forensic cases

      • micro-satellites

      • coupled with sequencing

  • CRISPER - Cas genome editing

    • clustered regularly interspaced short palindromic repeats

    • evolved as immunity in bacteria and archaea

      • cut foreign DNA

      • insert it into the host genome

      • memory built for next infection; better able to recognize the virus

      • cut and destroy foreign DNA

    • introduce Cas9 & guide RNA

    • effector complex binds target DNA

    • double stranded break

    • homologous recombination with donor DNA (repair)

    • non-homologous end joining (disrupt)

    • pros

      • long guide RNA provides specific targeting

      • used in intact cells

      • use alternate PAM sites (CAS from diff. species)

    • challenges

      • large protein

      • hard to place in living tissue