1. Introduction to Techniques
Restriction Enzymes, PCR and Gel Electrophoresis

2. Restriction Enzymes
Restriction enzymes are also called restriction endonucleases
They cut double stranded DNA at sequence specific sites
They can produce “sticky ends” or blunt ends depending on the enzyme
Sticky Ends
Blunt Ends
Sticky Ends
Blunt Ends

3. Restriction Enzymes
1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases
Restriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages)
The recognition sites are usually 4-12 nucleotides long
Sequences are palindromic (GAATTC)
There are hundreds of restriction enzymes currently being used

4. Restriction Enzymes: Applications
Restriction enzymes are commonly used in laboratories to create recombinant DNA
Harvest DNA products for other applications
DNase a general nuclease used to eliminate DNA in RNA samples

5. Restriction Enzymes
What is better for making recombinant DNA: Sticky ends or blunt ends?

6. Restriction Enzymes

7. Restriction Enzymes

8. Restriction Enzymes Student activity Potential Problems:
Wrong buffer
* activity
Online resources
Click on Techniques
Restriction enzymes are found in cutting and pasting

9. Gel Electrophoresis
Gel electrophoresis is used to separate nucleic acids (DNA and RNA) or proteins for analytical use
DNA and RNA are separated using agarose
Proteins are separated using polyacrylamide
The gel is a matrix (cross-linked polymers) that allow products to be separated
Separation is based on the size (not shape) of a product as it moves through a charged field
What is the charge on DNA and proteins? What is the shape? Why is this important?
How would you connect the electrodes?

10. Gel Electrophoresis
The negative charge is at the top (closest to the samples) and the positive charge is at the bottom
Samples are negatively charged and will travel towards the positive charge
DNA and RNA are negative because of their sugar-phosphate backbone
Proteins are denatured to give a constant shape and given a charge through the negative loading buffer used
Samples are diluted in a loading buffer that helps the samples stay in the wells

11. Gel Electrophoresis Applications Separating restriction digests
Analyzing/purifying PCR products
Sequencing
Protein analysis

12. Gel Electrophoresis

13. Gel Electrophoresis

14. Gel Electrophoresis
Sample agarose gel stained with ethidium bromide (EtBr)

15. Gel Electrophoresis Student activity
Practice loading a gel with 20uL Kool Aid or food coloring
Run gel and see color separation
Discuss what it means for the colors to separate

16. Gel Electrophoresis

17. Gel Electrophoresis

18. Gel Electrophoresis Online Resources: http://www.dnai.org/b/index.html
Potential Problems
Connecting the charges backward
Not enough loading dye
Running the gel too hot
Handling EtBr
Online Resources:
Click on Techniques
Gel electrophoresis is found in sorting and sequencing

19. PCR
Invented in 1983 by Kary Mullis (Nobel Prize in 1993 for its discovery)
Uses primers to exponentially amplify a specific region of DNA
Components needed for the reaction:
DNA
Primers to region of interest
DNA polymerase (Taq – used to synthesize the DNA)
dNTPS (the building blocks of the copied DNA)
Buffer (with appropriate salts to ensure the enzyme works properly)

20. PCR Three steps of the reaction:
Denaturation: High heat (94-98o) to separate the strands of DNA
Annealing: (50-60o – depends on the primers) this step allows the primers to bind to the denatured DNA strands
Elongation (74o) – DNA polymerase synthesizes the new strand
This step is dependant on the length of the product to be amplified (1min/1kb of DNA)
Check products with gel electrophoresis and sequencing

21. PCR: Cycles

22. PCR:

23. PCR: Thermocycler

24. PCR: Applications Used to test for gene products for disease diagnosis
Used to amplify small amounts of material
Forensics
Fossil Records
Used for recombinant DNA technology
Used for virus detection

25. Resources Potential Problems
No amplification due to wrong buffer conditions
No amplification due to lost enzyme activity
Primers are wrong
Online Resources:
Click on Techniques
PCR is found in amplifying

26. Online Resources