1. Biotechnology: Part 1 DNA Cloning, Restriction Enzymes and Plasmids
DNA Technology and Genomics

2. Biotechnology
DNA technology has launched a revolution in the area of biotechnology
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 specific DNA sequences, called restriction sites

5. NOT all bacteria have restriction enzymes, many do!
Where are restriction enzymes found?
In bacterial cells help fight viruses/foreign DNA
What is a restriction site?
Area on DNA that 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: phage) enters bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces.
NOT all bacteria have restriction enzymes, many do!

6. NON-BACTERIAL DNA
BACTERIAL DNA

7. Restriction Sites
Many different kinds of restriction enzymes with different names
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
Produce fragments with “sticky ends”
Can bond with complementary “sticky ends” of ANY other DNA fragment
DNA ligase (“molecular glue”)
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 Original plasmid (or phage) is called the cloning vector
DNA that carries foreign DNA into a cell and replicates in that cell
After insertion of a gene of interest into vector
Now vector is called Recombinant DNA

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

17. Steps in DNA Recombination (Cloning a Gene)
Isolate BOTH: 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)

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

20. 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

22. Plasmid Cloning

23. Identifying the gene of interest
How would you identify different genes?
Use different NUCLEIC ACID probes

24. 2 ways we know cell carries 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!