06 PCR

PCR

  • DNA polymerase requires primer

  • variant, quantitative

  • used for small amount of DNA curculating
  • HIV: RNA. Use reverse transcriptase to make copy of virus RNA: cDNA

Blotting

  • binding = hybridization

  • put a piece of filter paper on top. DNA stick to it

  • DNA left complementary to probe

  • for characterizing genes
  • length of fragments unique to each particular gene
  • Restriction nuclease added to Gene A and Gene B: breaking up into different fragments
  • Compare unknown gene that break up to gene A or B

  • determine genotype

  • same technique for RNA
  • studies gene expression by finding mRNA corresponding to standard

  • use antibody to protein instead of probe

Flow Cytometry

  • side and forward scatter
  • forward scatter based on cell size
  • side scatter based on granularity

  • lymphocytes: small, little granules
  • granulocytes: big, lots of granules

  • determine Hb F in maternal circulation = hemorrhage

Elisa

  • good for detect proteins

  • add serum to one of the wells
  • plate designed so antigen stick to surface
  • wash away serum and left with antigens bound

  • direct: for every antigen test for, must have an antibody enzyme linked
  • indirect: more common

  • unbound antibody: free antibody from last slide

  • any HIV antibody in sample will bind to antigen in plate

Microassays

FISH

  • like microarray, uses fluorescent

  • tell that gene is present on that chromosome