Chapter 9:Genetic Engineering

Section 9-2:

Manipulating DNA

Tools of DNA Manipulation

Biologists have tools to cut, separate, and read DNA sequences, and splice together those sequences in almost any order

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

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

EcoRI

Other Restriction Enzymes

** Note that the restriction enzyme recognition sequences are always palindromes.

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

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)

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

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

Reading DNA

After electrophoresis, gel has a pattern of bands that reveals the DNA sequence

Done by computers Used in Human Genome Project (handout)

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

Recombinant DNA

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

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

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

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