Chapter 9: Genetic Engineering

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 http://www.eng.auburn.edu/~yylee/che595/Reading%20Assignments/Recombinant%20DNA.htm

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