chapter 13 an introduction to cloning and recombinant dna powerpoints/chapter… · •fertilized...
TRANSCRIPT
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Chapter 13An Introduction to Cloning and
Recombinant DNA
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Clones
• Genetically identical organisms or moleculesderived from a common ancestor
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Cloning Plants from SingleCells
Fig. 13.1
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Cloning Animals
• Animals were cloned more than 20years ago
• Two techniques– Embryo splitting– Nuclear transfer
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library.thinkquest.org
animalscience.ucdavis.edu
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Embryo Splitting• Egg collected• Fertilized by in vitro fertilization (IVF)• Embryo is grown to 8–16 cells• Cells are separated• Separated cells grown into separate embryos• Embryos transplanted into surrogate mothers• May be used to clone any mammalian embryos,
including humans
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www.biotechnologyonline.gov.au
Cloning by nuclear transfer
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www.pnas.org
Cloning by nuclear transfer
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Nuclear Transfer• First done in 1986• More difficult• Nucleus is removed from an egg• Enucleated eggs are fused with other
cells• Embryos are transplanted into a
surrogate mother• In 1997, Dolly the sheep was the first
mammalian clone from an adult donor cell
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Cloned animals
Second addition
Second chance
at Texas A&M
Also cloned animals about to go extinct - gaur etc
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Cloning Mice byInjection of
Nuclei from AdultCells
Fig. 13.5
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Problems -
don’t live as long
not carbon copies/identical
develop diseases early
very low success rate - 0.1 - 3%
Dedifferentiation/reprogramming may not be completeor accurate
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Gene Cloning
All identical to starting gene - CLONES
Gene
Host
Cloning vector Recombinant DNA
Started with: few copies
GOAL: To get enough copies of the gene to manipulate
Multiply
Ended with: Many copies.
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Restriction enzymes
Nobel Prize
Werner Arber, Daniel Nathans and Hamilton O. Smith
1978
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Restriction Enzymes
Fig. 13.6
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GCCTAG
GATCC
GGATCC
G
G
CCTAG
Inserting foreign DNA using restrictionenzymes
GATCCG
G
CCTAG
BamHI BamHI
Ligase
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Frequency of occurrence of restriction sites
If DNA sequence has equal amounts of each base
If bases are distributed randomly
6 base cutter (1/4)6 = 1 site in ~4000 bp
4 base cutter (1/4)4 = 1 site in 256 bp
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Common Restriction Enzymes
Fig. 13.8
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Fig. 13.11a-d
Cloning DNA in Plasmid Vectors
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Fig. 13.11e-g
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Plasmid Usedto Carry DNAFragments
= Vectors
Fig. 13.10
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Table 19.2
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EcoRI EcoRI EcoRI EcoRI
4.0 kb 2.0 kb 3.0 kb
Problem: How to get the 2.0 kb piece to subclone into vector
Randomly Isolate specific fragmentShotgun cloning
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Steps in cloning a single piece of DNA
1. Appropriate restriction sites
2. Cut vector and foreign DNA with RE
3. Run on gel to separate fragments
4. Isolate specific fragment
5. Ligate with cut vector
6. Transform host bacteria. Selection.
7. Grow up colonies.
8. Isolate plasmid DNA.
9. Cut with RE to confirm presence of foreign DNA.
10. Run on gel to identify recombinant plasmids.
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Gel electrophoresis
-ve
+ve
Size separation
4.0 kb
3.0 kb
2.0 kb
5.0
3.5
2.8
2.4
1.5
2.1
Log
(kb)
Distance migrated
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Gel electrophoresis system or “gel box”
gel stained with ethidium bromide
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UV illumination of stained DNA fragments separated in an agarose gel by electrophoresis.
Credit: © Michael Gabridge/Visuals Unlimited 34173
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Selecting Cells with Vectors Vectors carry antibiotic resistance genes
Growing antibiotic-sensitive cells on media withantibiotics ensures that all growing cells mustcarry the vector
Selecting Cells with Recombinant Vectors While inserting the donor DNA, an existing gene
in the vector is inactivated
OR
In addition to the Donor gene a marker gene isadded
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Original vector - 4 kb with one RE (EcoRI) site
DNA to be inserted - 2 kb, flanked by same RE
Cut plasmids isolated from colonies. Run gel
Vector alone (no insert) - 1 band 4 kb
How to tell that plasmid now contains insert
Vector + insert - 2 bands 4 kb AND 2 kb