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
o DNA is cut in a staggered manner called “sticky ends”
o 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 length polymorphisms (RFLP's)
 Treat DNA with restriction enzymes and use gel electrophoresis to separate the restriction fragments
 DNA molecules can be identified by specific band patterns
o separated by size and electric charge
o DNA has + charge due to phosphates in backbone
o Smaller fragments move farther
o More voltage-move faster
Can also be used to isolate and purify genes for recombination (cut out bands in gel and remove DNA)
Usefulness:
o location of disease genes (linkage to known RFLP's)
o genetic fingerprint of individuals used in forensics
o diagnosis of Disease
o Human Gene Therapy
o vaccines
o pharmaceutical Products
o forensics
o agricultural Uses
o analysis of ancient DNA (woolly mammoth)
o prenatal diagnosis
o viral genetic detection (HIV)
o 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 enzyme cuts
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, cloned the gene for Human Insulin.
o In 1982, "Humulin" or Recombinant Human Insulin became the
first recombinant DNA drug approved by FDA.
o Today, Humulin is made in gigantic fermentation vats, 4 stories high and
filled with bacteria that operate 24 hours a day, year round.
o 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 polymerase molecules
Solution is heated to separate DNA helix
Then solution is cooled and the primers attach to their complementary sequences
DNA polymerase molecules recognize the primers and add nucleotides to primers
Both strands of 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 more than 40min.
PCR is an incredibly versatile technique:
Can now “pull out” a piece of DNA sequence, like a gene, from a larger collection of DNA
like the whole cellular genome.
Just define the gene with “flanking” primers and get a lot of copies 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.