1. Chapter 9: Genetic Engineering
Section 9-2:
Manipulating DNA

2. Tools of DNA Manipulation
Biologists have tools to cut, separate, and read DNA sequences, and splice together those sequences in almost any order

3. Tool #1: Cutting DNA
Restriction enzymes are proteins that cut DNA at specific sequences
Each RE recognizes a different sequence – there are more than 100
DNA can be cut into smaller, precisely sized fragments allowing scientists to work with a few hundred nucleotides at a time

4. Restriction Enzymes
When RE cut DNA they can leave blunt ends or sticky ends
Sticky ends are single-stranded regions on either side of the cut
This is an example using EcoRI
This is an example using SmaI

5. EcoRI

6. Other Restriction Enzymes
** Note that the restriction enzyme recognition sequences are always palindromes.

7. Tool #2: Separating DNA
Electrophoresis is a technique used to separate DNA fragments cut by restriction enzymes.
Fragments move through a special gel made of agarose

8. Electrophoresis Step 1: Cut DNA using RE
Step 2: Place fragments at one of the gel in wells
Step 3: Apply electric current. (Set gel into buffer solution that conducts current – Negative electrode at the end with the DNA fragments)

9. Electrophoresis
DNA has a negative charge, so the current will pull the DNA fragments toward the positive electrode
Smaller fragments move through the gel faster than larger fragments

10. Tool #3: Reading DNA
Once REs have cut a sample of DNA, fragments can be placed in a test tube with DNA polymerase and nucleotides to “read” the fragments
Complementary DNA strand produced using chemically modified nucleotides that stop assembly at certain spots – fragments then separated by electrophoresis

11. Reading DNA
After electrophoresis, gel has a pattern of bands that reveals the DNA sequence
Done by computers
Used in Human Genome Project (handout)

12. Tool #4: Splicing DNA Sticky ends left by some REs
If two samples of DNA are cut with the same RE, their sticky ends can be matched up and enzymes can be used to permanently join the fragments
Newly joined pieces of DNA are called recombinant DNA

13. Recombinant DNA

14. Cell Transformation
Involves inserting new genes into a cell, changing the cell’s genetic makeup
Uses recombinant DNA
Can be done in prokaryotes and eukaryotes

15. Transforming Bacteria
Some bacteria have their regular DNA plus a small, circular, extra piece of DNA called a plasmid
Plasmid can be made recombinant using REs – new genes are spliced in
Recombinant plasmids are mixed into bacterial cultures - under the right conditions they will be picked up by some bacteria
These bacteria will then reproduce more bacteria containing the recombinant plasmid

16. Transforming Eukaryotes
More difficult to get a eukaryote to accept foreign DNA because they are more complex
Yeasts (eukaryote) contain plasmids like bacteria, therefore are commonly used for transformation
Animal and plant cells without plasmids have been transformed by injecting new DNA