DNA SEQUENCING

1. DNA FRAGMENTS DIVIDED BY SIZE
2. FRAGMENT SELECTED FOR SEQUENCING USING THE
   TECHNIQUE OF FREDRICK SANGER
       a. SOLUTION CONTAINING DNA DIVIDED INTO FOUR
      TEST TUBES
       b. EACH TEST TUBE CONTAINS A DIFFERENT
      RADIOACTIVE NUCLEOTIDE (A,T,C,G)
       c. DNA POLYMERASE ADDED TO EACH TUBE ADDS BOTH
      RADIOACTIVE AND NON-RADIOACTIVE
NUCLEOTIDES
       d. WHEN A RADIOACTIVE NUCLEOTIDE IS ADDED
      STRAND GROWTH STOPS
       e. DNA FROM EACH TUBE IS PUT ON A GEL AND
      SUBJECTED TO
ELECTROPHORESIS
       f. THE GEL IS PLACED ON XRAY FILM AND THE FILM
      EXPOSED

KNOWING THE DNA SEQUENCE OF SOMETHING ALLOWS PROBES TO BE MADE

PROBES

PROBES ARE SHORT DNA SEQUENCES SYNTHESIZED FROM RADIOACTIVELY LABELLED NUCLEOTIDES THAT ARE ENGINEERED TO RECOGNIZE PART OF THE DNA OF INTEREST

WHEN A PROBE PAIRS WITH THE DNA OF INTEREST, NUCLEIC ACID HYBRIDIZATION OCCURS

PROBES ARE USED TO LOCATE TRANSFORMED CELLS

PROCEDURE:

1. CELLS ARE TRANSFORMED WITH PLASMIDS THAT CARRY
   THE GENE OF INTEREST AND A GENE FOR RESISTANCE
   TO AN ANTIBIOTIC
2. CELLS ARE GROWN ON AGAR CONTAINING AN
   ANTIBIOTIC
       a. ONLY CELLS THAT HAVE BEEN TRANSFORMED
      SURVIVE
       b. COLONIES GROW
3. COLONIES BLOTTED ON NITROCELLULOSE FILTERS
       a. CELLS ON FILTERS RUPTURED
       b. DNA "FIXED" TO FILTER
       c. DNA STRAND UNWOUND
       d. PROBE ADDED TO FILTER
       e. PROBE BINDS TO DNA OF A COMPLIMENTARY
      SEQUENCE
       f. FILTER PUT ON XRAY FILM AND TRANSFORMED
      COLONIES EXPOSE
FILM
       g. TRANSFORMED COLONIES IDENTIFIED cDNA
      (COMPLIMENTARY DNA)

cDNA IS A STRAND OF DNA MADE FROM A STRAND OF mRNA

mRNA IS ISOLATED FROM A TISSUE AND COMBINED WITH AN ENZYME CALLED REVERSE TRANSCRIPTASE AND NUCLEOTIDES TO MAKE A STRAND OF DNA

THIS STRAND OF DNA CAN BE PUT INTO A PLASMID TO CREATE A DNA LIBRARY

ENETICALLY ENGINEERED BACTERIA

1. BACTERIA ENGINEERED TO PRODUCE:
       a. INSULIN
       b. HEMOGLOBIN
       c. INTERFERON
       d. ALBUMIN
2. ENGINEERED BACTERIA:
       a. DEGRADE OIL
       b. MANUFACTURE ALCOHOL
       c. LEAVE CROP PLANTS ALONE
       d. MAKE CROPS FROST RESISTANT

GENETICALLY ENGINEERED PLANTS

1. BENEFICIALLY GENES ARE SOUGHT AFTER IN LITTLE
   KNOWN PLANTS
       a. THESE GENES MAY NOT HAVE BEEN DISCOVERED YET
2. PLANTS GROWN IN TISSUE CULTURE MAY MUTATE TO
   FORM NEW STRAINS OF PLANTS THAT MAY BE ABLE TO
       WITHSTAND ADVERSE CONDITIONS
3. METHODS OF GENE TRANSFER
       a. DNA FRAGMENTS INSERTED INTO A Ti PLASMID
      FROM THE
BACTERIUM, AGROBACTERIUM
               TUMEFACIENS WHICH INFECTS MANY PLANTS.

DNA FROM THE BACTERIA ENTERS THE PLANT DNA AND IS
INCORPORATED INTO THE DNA

PROBLEM: ONLY BEANS, PEAS,POTATOES AND OTHER DICOTS MONOCOTS AND SOME DICOTS ARE TRANSFORMED BY ELECTRIC SHOCK OR BY "BULLETS" IN A PISTOL

GENETIC ENGINEERING OF ANIMALS

1. SOMATOTROPISM AND RATS
       a. TOO LITTLE SOMATOTROPIN LEADS TO DWARFISM
       b. GENE FOR SOMATOTROPIN IS INJECTED INTO
      MOUSE EMBRYOS
       c. MICE THAT DEVELOPE ARE 1 AND 1/2 TIMES
      LARGER THAN THOSE NOT RECEIVING THE GENE
2. POSSIBLE USES INCLUDE:
       a. CLONING OF GENETICALLY SUPERIOR ANIMALS
       b. PROVIDING TISSUS AND ORGANS FOR MEDICAL
      USES AND RESEARCH