1. Chapter 20: Part 1 DNA Cloning and Plasmids
DNA Technology and Genomics

2. Biotechnology
DNA technology has launched a revolution in the area of biotechnology
The manipulation of organisms or their genetic components to make useful products
Example: gene cloning
Usually uses bacterial plasmids

3. Figure 20.2
Bacterium
Bacterial chromosome
Plasmid
Cell containing gene of interest
Recombinant DNA (plasmid)
Gene of interest
DNA of chromosome
Recombinant bacterium
Protein harvested
Basic research on protein
Copies of gene
Basic research on gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
Protein expressed by gene of interest 3
Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

4. How do You Make Recombinant DNA?
Use bacterial restriction enzymes
Called restriction endonucleases
Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

5. Restriction enzymes Where are restriction enzymes found?
In bacterial cells, to help fight viruses/ foreign DNA
What is a restriction site?
The area on DNA that the restriction enzyme recognizes
Every time this DNA sequence occurs in bacterium’s own DNA, methyl groups (-CH3) are added  prevents restriction enzyme from working.
When foreign DNA (ex: a bacterial virus) enters bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces.
NOT all bacteria have restriction enzymes, many do!
But there is a wide variety of restriction enzymes that have been discovered and continue to be discovered

7. Restriction Sites Many different kinds
EcoRI, BamHI, HindIII, HaeIII, etc.
Restriction site is like a palindrome
A palindrome is a word or phrase which reads the same in both directions.
These enzymes make “blunt” and “sticky” ends.
What is the difference?

8. Restriction Enzymes Enzymes usually make many cuts in a DNA molecule
Yields a set of restriction fragments
Most useful restriction enzymes cut DNA in a staggered way
Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of any other DNA fragment
DNA ligase is an enzyme that seals the bonds between restriction fragments

10. Different restriction enzymes that can be used

12. Restriction Enzymes
ANIMATION #1

13. Restriction Enzymes: Proteins that cut the DNA in a specific place
Recombinant Plasmid

14. Transformation  bacteria takes in plasmid from environment

15. Cloning The original plasmid is called the cloning vector
DNA molecule that can carry foreign DNA into a cell and replicates there
After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA
Recombinant Plasmid

16. How do you clone a gene using a plasmid?

17. Steps in DNA recombination
Isolate “needed: DNA gene & plasmid
Cut (“digest”) BOTH DNA samples (gene of interest and plasmid) with the SAME restriction enzyme
Mix DNA’s together- they join at sticky ends via DNA base pairing
Add ligase to seals up sticky ends
**Product is the recombinant DNA
Add CaCl2 & bacteria (makes cell competent)
Collect products (proteins) of cloned gene  (Ex: insulin, Human Growth Hormone)

19. Steps in Cloning a Gene Using a Plasmid
ANIMATION #8

21. Why make DNA recombinants?
There are 3 main reasons for creating recombinant DNA
to create a protein product
to create multiple copies of genes
to insert foreign genes into other organisms to give those organisms a new trait
Make DNA fingerprints
Recombinant DNA is used widely today to create large amounts of protein for treating illnesses
Ex: In 1982, insulin became the first recombinant DNA drug to hit the market

23. Plasmid Cloning

25. Identifying the gene of interest
How would you identify different genes?
Use different probes

26. 2 ways that we know the cell clones carry the recombinant plasmids?
Look for the specific product (protein) that the foreign DNA codes for
Is the bacteria making this protein?
If yes, the cloning worked!
Use a “nucleic acid probe”
A single stranded nucleic acid molecule used to “tag” a specific nucleotide sequence in a sample
Hydrogen bonding is used!