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Chapter 9: Genetic Engineering

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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

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