DNA TECHNOLOGY-Chapter 20

RESTRICTION ENDONUCLEASES (Restriction enzymes)

  • cut foreign DNA into small segments
  • occur naturally in bacteria
  • protection from the DNA of other organisms
  • Different enzymes recognize and cut different nucleotide sequences (many are palindromes)
  • Named for bacteria they come from:

EX: EcoR1; HindIII; BamH1

  • Can be used to combine DNA from different organisms
  • DNA is cut in a staggered manner called “sticky ends”
  • DNA pieces cut with same restriction enzymes can be joined by DNA ligase

REVERSE TRANSCRIPTASE

  • Enzyme from RETROVIRUSES that transcribes an RNA template into DNA
  • info flows backwards RNA → DNA
  • Can be used to put eukaryotic genes into bacteria
  • Bacteria don’t process DNA so eukaryotic genes with introns can’t be used directly
  • Reverse transcriptase enzymes can take a message with introns removed and change it into a gene

RFLP’s (Restriction fragment length polymorphisms

  • DNA segments with different alleles of a gene result in different banding patterns
  • the restriction fragment length differences = restriction fragment lengthpolymorphisms (RFLP's)
  • Treat DNA with restriction enzymes and use gel electrophoresis to separatethe restriction fragments
  • DNA molecules can be identified by specific band patterns
  • separated by size and electric charge
  • DNA has + charge due to phosphates in backbone
  • Smaller fragments move farther
  • More voltage-move faster

Can also be used to isolate and purify genes for recombination (cut out bands in gel and remove DNA)

Usefulness:

  • location of disease genes (linkage to known RFLP's)
  • genetic fingerprint of individuals used in forensics
  • diagnosis of Disease
  • Human Gene Therapy
  • vaccines
  • pharmaceutical Products
  • forensics
  • agricultural Uses
  • analysis of ancient DNA (woolly mammoth)
  • prenatal diagnosis
  • viral genetic detection (HIV)
  • human genome project

PLASMIDS
Small circular self replicating DNA molecule in bacteria separate from bacterial chromosome

2-30 genes

Often carry genes for antibiotic resistance or genetic recombination

Can be exchanged between bacteria

Bacterial “sex” = conjugation (facilitated by F plasmids)
Role in rapid evolution
Method for spreading “antibiotic resistance”

Can be cut with restriction enzymes and used to incorporate foreign DNA into bacteria

Bacteria reproduce, copying the inserted gene along with plasmid

LAB 6: Cells can be made “competent” by using calcium chloride and “heat shock” to change their cell walls
- makes them better able to pick up plasmids; rapidly growing cells are made competent more easily

Gene-Cloning Steps:

  • Isolation of two kinds of DNA (i.e., bacterial and foreign DNA)
  • Treatment of the plasmid and foreign DNA with the same restriction enzymecuts both at the same restriction site
  • Mixture of foreign DNA with clipped plasmids
  • Sticky ends join
  • DNA ligase seals connections to form recombinant DNA molecule
  • introduction of recombinant plasmid into bacterial calls (via transformation)
  • production of multiple gene copies by gene cloning;
  • bacteria reproduce, cloning the inserted gene in the process

Ex: 1978-scientists at Genentech, clonedthe gene for Human Insulin.

  • In 1982, "Humulin" or Recombinant Human Insulin became the

first recombinant DNA drug approved by FDA.

  • Today, Humulin is made in gigantic fermentation vats, 4 stories high and

filled with bacteria that operate 24 hours a day,year round.

  • The human insulin protein made by the E. coli bacteria is

collected from the vats, purified, and packaged for use by diabetic patients

GFP (Green fluorescent protein)

Fluorescent pigment found in jellyfish

Used to determine whether a foreign gene had been inserted into an organism’s genome

Way to track “invisible genes”

PCR (Polymerase chain reaction)

Can make billions of copies of a piece of DNA (amplification) in vitro

DNA is incubated with special primers and DNA polymerasemolecules

Solution is heated to separate DNA helix

Then solution is cooled and the primers attachto their complementary sequences

DNA polymerase molecules recognize theprimers and add nucleotides to primers

Both strandsof DNA are copied; then copied strands are used as templates in next round

Heating/cooling process is repeated many times to get many copies

DNA Polymerase from archaebacteria Thermus aquaticus is used (Taq)

Highly thermostable – withstands temperatures up to 95°C for morethan 40min.

PCR is an incredibly versatile technique:

Can now “pull out” a piece of DNAsequence, like a gene, from a larger collection of DNA

like the wholecellular genome.

Just define the gene with “flanking” primers and get a lot ofcopies in 40 minutes

Can also add in a restriction site to the copies of the gene(if one doesn’t exist)
by adding them at the end of the original primers.