Mapping and Sequencing the Human Genome by HC120717021823

VIEWS: 16 PAGES: 43

									Cloning and Expression of Genes
• Insulin: Harmone regulating blood sugar
  (glucose)
• Obtain Insulin Gene from Human DNA
• Insert gene in bacterial cells
• Select cells that have desired gene
• Induce bacteria to express foreign gene to
  produce insulin
• Collect and purify insulin
          Isolating the Gene
• Isolate mRNA
• Copy to DNA with reverse transcriptase
• cDNA (copy or complementary DNA)
          Inserting the Gene
• Carrier DNA is a vector
• Plasmids: Circular DNA found in
  microorganisms
• Cloning Vector
• Easily transferred, incorporated, and
  isolated
• Inserted with restriction enzymes
• Cut up foreign DNA
   Selecting Cells with Desired
              Gene
• Most cells don’t take up DNA
• Ampicillin resistance gene in plasmid
  vectors
• Only cells with plasmids grow
• Blue-White Screening for gene or not
• Intact bacteria gene or not, Blue or White
Isolating the Insulin
            Analysis of DNA
•   Gel Electrophoresis
•   DNA Sequencing
•   Hybridization
•   Polymerase Chain Reaction
•   DNA Chips
•   Human Genome Project
                    DNA LIGATION



             

        ’    ’                   



                              
                                       ’   ’
        ’    ’

Ligase Joins 5' phosphate
     to 3' hydroxyl
          Gel Electrophoresis
• Sort DNA by Size
• Electrophoresis is separation of particles in an
  electric field based on charge
• Thin Agarose Gels: Matrix of interlocking fibers
• DNA has negative charge
• Repates through matrix
• Ethidium Intercalator for visualization
• Entanglement and speed related to charge and size
• Restriction Map: Different restriction enzymes,
  different size maps, ID restriction sites
GEL ELECTROPHORESIS - SIZE SORTING

           Electrode   Samples




                       Slower
                                 Gel
  Buffer
                                 Electrode
          RESTRICTION ENDONUCLEASES

 EcoRI




HindIII




   AluI




HaeIII
             Sequencing
• Isolate fragment to be sequenced as SS
• 4 Tubes, 4 Bases
• Add mix of nucleotides, DNA polymerase,
  and primers with radioactive labels
• In each tube as single base is modified
  (dideoxynucleotide)
• Once added no other nucleotides added
• Chain Termination Sequencing
DNA Sequencing
             Hybridization
• Binding of complementary DNA strands to
  each other
• Find insulin sequence? Need prior
  information
• Hybridize with part of the insulin sequence
             Gene Expression
•   Study production of mRNA in the cell
•   Clone genes of interest
•   Make cDNA probes
•   Sample mRNA populations of cell
•   Detect by hybridization to radioactive probe
    Polymerase Chain Reaction
• Amplify a single piece of DNA
• Makes a large number of copies
• Target specific primers allow specific
  sequences to be amplified
DNA Polymerase
POLYMERASE
   CHAIN
 REACTION
               DNA Arrays
•   DNA Sequences Affixed to Solid Support
•   Genotyping and Gene Expression
•   Entire Genomes on Chips
•   Look at Relative Intensity Levels
•   Massively Parallel
•   Loads of Data
•   Interpretation, Storage, Analysis?
http://www.affymetrix.com
http://cmgm.stanford.edu/pbrown/
                            ANTIBODY AFFINITY

                                                       CACCATGTGAC

     Add oligo with
      Biotin label
                                        +
                                  GTGGTACACTG    B

                                             Anneal
      Heat and cool

                                                     CACCATGTGAC
         Add
Paramagnetic-Streptavidin               +            GTGGTACACTG     B
       Particles
                                            PMP




                                                Bind


   Isolate with Magnet                    CACCATGTGAC
                                                                               N

                                            GTGGTACACTG     B            PMP

                                                                               S
   Mapping and Sequencing the
        Human Genome
• Find all genes in Human Genome
• Develop Tools for Analysis in Biology and
  Medicine
• Tools:
  – Restriction Enzymes
  – PCR
  – DNA Arrays

								
To top