BIOTECHNOLOGY BIOTECHNOLOGY RECOMBINANT DNA RECOMBINANT DNA TECHNOLOGY TECHNOLOGY GENETIC ENGINEERING GENETIC ENGINEERING

emryologistromanianBiotechnology

Oct 22, 2013 (3 years and 11 months ago)

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BIOTECHNOLOGY
BIOTECHNOLOGY
RECOMBINANT DNA
RECOMBINANT DNA
TECHNOLOGY
TECHNOLOGY
GENETIC ENGINEERING
GENETIC ENGINEERING
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GENETIC ENGINEERING -
RECOMBINANT
DNA TECHNOL
OG
Y BIOTECHNOLOGY
PURIFICATION OF DNA (Chromosomal and plasmid)
RESTRICTION ENZYMES AND CLONING
Restriction/Modification Foreign/Self
Hemophilus influenzae
HincII restriction enzyme
Escherichia coli
strain R1 EcoRI restriction enzyme
Specific sites, palindromes
Hybrid molecules constructed in vitro, DNA ligase
Vector, Target (or foreign) DNA, Transformation
Cloning
DNA NUCLEOTIDE SEQUENCE DETERMINATION
GENE LIBRARY
Eukaryotes, cDNA clone
POLYMERASE CHAIN REACTION (PCR)
Denature DNA, anneal primers, polymerize with DNA
polymerization enzyme, start over
Exponential amplification
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GENETIC ENGINEERING TECHNIQUES
1.
TRANSFORMATION BY PLASMIDS
2.
CUTTING DNA AT SPECIFIC SEQUENCES; RESTRICTION
ENZYMES CUT SPECIFIC SEQUENCES, CALLED "SITES"
RESTRICTION / MODIFICATION NOBEL
3.
MOLECULAR CLONING
FOREIGN DNA INTO VECTORS
GENERATES HYBRID DNA MOLECULES NOBEL
4.
DETERMINATION OF DNA SEQUENCE NOBEL
5.
SYNTHESIS OF DNA STRANDS OF PREDETERMINED
SEQUENCE
6.
POLYMERASE CHAIN REACTION-
AMPLIFY "ANY" SPECIFIC DNA FRAGMENT OR GENE
NOBEL
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RESTRICTION / MODIFICATION
RESTRICTION -
RECOGNITION OF FOREIGN DNA
NON-SELF AND CLEAVAGE BY RESTRICTION
ENZYME [AT SPECIFIC NUCLEOTIDE SEQUENCES
OF 4 TO 6 BAS
E PAIRS]
MODIFICATION -
CHEMICAL MODIFICATION OF
"OWN" DNA [THAT IS, DNA SYNTHESIZED WITHIN
"OWN" CELL] SO IT CAN NOT BE CLEAVED BY
RESTRICTION ENZYME
MODIFICATION IS ADDITION OF ME
THYL GROUP
TO NUCLEIC ACID BASE BY MODIFICATION
ENZYME [DNA METHYLASE]
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RESTRICTION ENZYME HincII
HEMOPHILUS INFLUENZAE
EXTRACT PLUS:
E. COLI
PHAGE T7 DNA > CUT INTO 40 SPECIFIC FRAGMENTS
H. INFLUENZAE
DNA > NO CUTTING
CONCLUDE: ENZYME IN EXTRACT WHICH:
A.
RECOGNIZES AND CUTS FOREIGN DNA
B.
CLEAVES FOREIGN DNA AT SPECIFIC SEQUENCES
CALLED "SITES"
RESTRICTION ENZYMES "SITES" ARE NOT SITES IN A
PHYSIOLOGICAL SENSE. THEY ARE SIMPLY 4 TO 6
NUCLEOTIDES THAT HAPPEN TO BE PRESENT IN DNA
WITHIN GENES OR OUTSIDE GENES. WE CALL THEM
SITES FOR CONVENIENCE.
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HincII RESTRICTION ENZYME CUTS:
[ENDONUCLEASES]
SYMMETRY; PALINDROME
HincII
PLASMID
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ADENINE
N6 METHYL
ADENINE
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HincII
WILL NOT CUT
PLASMID MODIFIED AS "SELF" IN HEMOPHILUS
INFLUENZAE
CELLS
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E. COLI
RESTRICTION ENZYME EcoRI
"STICKY" ENDS
COMPLEMENTARY
"STICKY" ENDS
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CUTTING DNA AT SPECIFIC SITES
EcoRI
INACTIVATE EcoRI
COOL SLOWLY
REFORMING DNA
MOLECULES:
DNA LIGASE
GENES
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BamHI RESTRICTION ENZYME
BACILLUS AMYLOLIQUEFACIENS
STRAIN H
BamHI MODIFICATION ENZYME
N4 METHYL
CYTOSINE
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MOLECULAR
CLONING
1.
CONSTRUCT HYBRID DNA MOLECULE
TARGET -
GENE OR DNA FRAGMENT OF INTEREST
PLUS A VECTOR -
PLASMID, PHAGE, OR VIRUS
WHICH CARRIES THE GENE OF INTEREST
2.
INTRODUCE HYBRID DNA MOLECULES INTO HOST
CELLS [BACTERIA, YEAST, ANIMAL CELLS, OR
HUMAN CELLS] WHICH REPLICATE THE HYBRID
MOLECULES AND ALLOW EXPRESSION OF THE
TARGET GENE
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VECTOR
TARGET
GENE
EcoRI
SITE
EcoRI
SITE
EcoRI
SITE
PEN-RESISTANCE
ADD
EcoRI
ENZYME
EcoRI
[AND OTHER FRAGMENTS]
INACTIVATE EcoRI; MIX; ANNEAL; LIGATE
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TRANSFORM PENICILLIN-SENSITIVE
BACTERIA WITH MIXTURE
SELECT TRANSFORMANTS
e.g., PENICILLIN-RESISTANCE DUE TO VECTOR GENE
IDENTIFY TRANSFORMANTS WITH A GENE OR INTEREST (TARGET)
TARGET GENE EXPRESSED IN BACTERIUM
TARGET
PROTEIN
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INTRONS ARE PROBLEMS FOR CLONING HUMAN
[OR HIGHER ORGANISM] GENES. INTRONS ARE TOO
LONG TO PERMIT USE IN THE TEST TUBE.
1.
PURIFY MESSENGER RNA; [INTRONS ARE ALREADY
SPLICED OUT]
2.
COPY mRNA INTO DNA WITH REVERSE
TRANSCRIPTASE: USE THIS DNA AS TARGET
3.
CUT TARGET DNA WITH RESTRICTION ENZYME
4.
PURIFY VECTOR DNA AND CUT WITH RESTRICTION
ENZYME
5.
MIX THE DNA's. INACTIVATE THE RESTRICTION ENZYME,
ANNEAL, LIGATE
6.
TRANSFORM BACTERIAL HOST, SELECT
TRANSFORMANTS, IDENTIFY THOSE WITH HYBRID
PLASMID
THESE CONTAIN c-DNA CLONE, OR
COMPLEMENTARY DNA CLONE
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CLONING VECTORS SHOULD
HAVE SEVERAL PROPERTIES:
1.
THEY SHOULD BE SMALL, EASILY ISOLATED
MOLECULES
2.
THEY SHOULD HAVE ONLY ONE SITE FOR THE
RESTRICTION ENZYME TO BE USED
3.
THEY SHOULD HAVE THE RESTRICTION ENZYME SITE
IN SOME LOCATION OTHER THAN AN ESSENTIAL
GENE
4.
THEY SHOULD CARRY SOME SELECTABLE GENE OR
MARKER, LIKE TETRACYCLINE-RESISTANCE
5.
THEY MUST BE REPLICONS, THAT IS, CAPABLE OF
BEING REPLICATED WHEN THE HOST CELLS GROW
AND DIVIDE.
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cDNA CLONE
HUMAN mRNA [AFTER PROCESSING]
5'
3'
<
READI
NG FRAME
>
REVERSE TRANSCRIPTASE = R.T.
5'
3' RNA
3'
5' DNA
RNA DEGRADED
R.T.
READING FRAME
RESTRICTION ENZYME
cDNA CUT WITH RESTRICTION ENZYME
NO INTRONS PRESENT
cDNA CAN BE CLONED
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DETERMINING SEQUENCE OF DNA

CHEMICAL SYNTHESIS OF DNA OF DEFINED
[PRE-DETERMINED] SEQUENCE

POLYMERASE CHAIN REACTION = PCR
AMPLIFICATION
OF SPECIFIC DNA FRAGMENT
DENATURE 90-95oC
(SEPARATE STRANDS)
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ADD PRIMERS
[COMPLEMENTARY TO ENDS]
[HUGH EXCESS]
ANNEAL
3'
3'
POLYMERIZE COMPLEMENTARY STRANDS
DNA POLYMERASE
dATP, dGTP, dCTP, dTTP (72oC)
NEW SYNTHESIS
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FIRST CYCLE DOUBLED AMOUNT
OF DNA BETWEEN PRIMERS
20 -
30 CYCLES
106
-1
0
7
FOLD
AMPLIFICATION
~3 HRS
PRINCIPAL PRODUCT
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HIV PROVIRUS
HUMAN
CHROMOSOME
A
B
HIV DNA SPECIFIC
PRIMERS
DENATURE
ANNEAL PRIMERS
POLYMERIZE
30 CYCLES
5'
3'
3'
5'