Download DNA Technology

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Recombinant DNA Technology
I.
Natural recombination:
A.
Provides means of genetic exchange between closely related species:
Conjugation
Transformation
Transduction
B.
II.
III.
Recombinant DNA Technology:
A.
Methods used in genetic engineering:
Depend on universality of:
Genetic code
Mechanisms of protein synthesis
B.
Allows artificial recombination of DNA from very different organisms:
Plants
Bacteria
Animals
Yeast
Viruses
C.
Foreign DNA inserted into cell:
Cell then produces proteins coded for by the foreign genes
D.
Fairly recent aspect of bacterial genetics:
Started in the late 1960's:
Mechanisms used in recombinant DNA technology:
A.
Similar to processes that occur during natural recombination:
During both natural recombination and the production of recombinant DNA
DNA from two separate sources:
Fragmented by:
Normal cell enzymes
Joined by:
Normal cell enzymes
B.
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Genetic Engineering:
Deliberate modification of an organism's genetic information by directly
changing its DNA
Discovery of two types of enzymes allowed production of recombinant DNA:
Restriction endonucleases:
Cut double stranded DNA:
2
At specific nucleotide sequences
Normal function is to destroy phage DNA after its enterence into a host
cell
DNA ligase:
Enzyme:
Repairs breaks in the sugar-phosphate backbone of DNA
C.
Four major steps in producing recombinant DNA:
Step 1.
DNA for a particular phenotype
identified
Purified
Isolated
May use gene machine to:
Synthesize eukaryotic genes:
so that they can be expressed in prokaryotic cells:
DNA sequence of:
Human insulin
Human growth hormone
Synthesized from the amino acid sequence
of the proteins
Isolation actual genes from chromosomes:
Difficult to do with eukaryotic genes
Easily done with viral genes:
Gene for surface antigen of the Hepatitis B virus
isolated:
Introduced into bacterium:
Bacteria make the viral protein:
Protein used as vaccine for hepatitis B
Step 2.
Purified DNA fused with other pieces of DNA to form recombinant
DNA molecules:
Fragmented donor DNA attached to a vector:
Carrier of foreign genes:
Plasmids:
Contain no fertility factors
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Viruses
Vector DNA must be:
Able to replicate in host cell
Stable in the host cell
Restriction endonucleases and DNA ligase:
Enzymes used to insert foreign DNA into vector:
Restriction endonucleases:
Cut DNA into fragments:
Cut at specific nucleotide sequences:
Each restriction endonuclease recognizes a specific sequence:
Each cuts DNA at a different place
Fragmented donor DNA attached to a vector that has been:
Cut by the same restriction endonuclease
Break double stranded DNA in regions called palindromes:
Highly specific:
Each cleaves DNA at a specific palindrome
Protect cells from foreign DNA (viruses):
Palindrome:
Word, phrase, sentence, etc.
Reads the same backwards or forwards:
Radar
Otto
Eve
Deed
Able was I ere I saw Elba.
Madam I'm Adam.
A man, a plan, a canal, Panama.
Straw, no too stupid a fad, I put soot on
warts.
Results in formation of DNA fragments with sticky ends:
Sticky ends able to pair with any complementary sticky end
regardless of source:
────────────────────────── DNA
strand
T (A A T T) C C G C
A (T T A A ) G G C G
────────────────────────── DNA
strand
DNA molecule with palindrome. Restriction endonuclease
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cuts DNA between A and T on one strand and between T
and C on the other.
─────────────
─────────────
DNA strand
TA A T T
CC GC
A
──
DNA strand
TT A AGG CG
────────────────────
"sticky ends." Sticky ends will form hydrogen bonds with
any other DNA cleaved with the same enzyme.
DNA ligase:
Used to join fragments into a recombinant DNA molecule:
Repairs breaks in sugar phosphate backbone
Reseals cuts made by restriction endonuclease:
DNA from any source can be joined to DNA from any
other source
Step 3. Recombinant DNA molecules:
Inserted into a host cell
Vector moved into recipient (host) cells using transformation
techniques:
Electroporation:
Pulses of high-voltage electricity:
Increase membrane permeability
In E. Coli use:
Cold CaCl2 treatment
Increases membrane permeability
Step 4. Grown as a clone:
Recipient cell containing the desired foreign gene must be selected:
Use genetic markers:
Genes for:
Pigment production
Antibiotic resistance
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Use selective media
Necessary because:
Other genes may combine with the vector:
Taken up by recipient cells
Getting the recombined gene expressed:
Foreign gene must be expressed:
Recipient cell must produce large amounts of the desired
product
IV.
Potential benefits of genetic engineering:
A.
Synthesis of human hormones:
Insulin:
Human insulin gene introduced into E. coli:
Human insulin produced by E. coli used to treat diabetes
Human growth hormone
Human growth hormone gene introduced into E. coli:
Human growth hormone produced by E. coli used to treat children
with growth deficiencies
B.
Production of large amounts of antibiotics
Production of anti-cancer drugs:
Interferon
C.
Increase nitrogen fixing capabilities of plants
D.
Making animal proteins for food
E.
Adding photosynthetic genes to more kinds of microorganisms
F.
Genetic repair of hereditary defects
G.
Improving marketing qualities of fruits and vegetables
V.
Potential hazards of genetic engineering:
A.
Introduction of foreign DNA into bacteria could create potentially dangerous
strains of bacteria:
Done by accident or design:
In 1974 Geneticists called a:
Voluntary halt to recombinant studies to evaluate possible
risks:
In 1976:
The National Institutes of Health Guidelines for
Recombinant DNA research were approved:
1976 E. coli strain developed:
Grows only in laboratory environment:
Grows only at temperatures below 18oC
Will not grow in humans
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Has cell wall defects
Killed by sunlight
In 1982 Guidelines relaxed:
Hazards have not developed
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