Biotechnology Read textbook sections 20.1 & 20.2 on your own Draw 10 boxes to complete the following notes Turn into the purple box when you are done.

Genetic Engineering: Direct manipulation of Genes for practical Purposes

Gene: 1/100,000 of a chromosome

DNA Gene Non-coding Section of DNA Gene cloning: to be able to work with just the genes you want

Well, how do we clone pieces of DNA??? We use Plasmids!

A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. In nature, this information is often a gene that codes for antibiotic resistance or other “contingency” functions.

Plasmids can be used to transfer a gene of interest.

Box #1 What is genetic engineering? What is a plasmid?

But how???

1 st, let’s Look at how To cut up DNA

Restriction enzymes are enzymes that cut DNA at specific nucleotide sequences known as restriction sites.

Box #2 What is the function of restriction enzymes?

The Restriction Enzyme How does it Know where to cut? Restriction Site

Restriction Site Restriction sites – how often do they occur? Frequently! Why? Short Segment Therefore, you have Many restriction Fragments when you Cut up a piece of DNA

Box #3 What is the relationship between a restriction site and a restriction fragment?

Same Restriction Enzyme Sticky Ends

Recombinant DNA DNA from two different sources (same species or not)

Take DNA Out of Cell Take Plasmid Out of Bacteria Cut Human DNA With Restriction Enzyme Cut Bactera Plasmid (DNA) With Same Restriction Enzyme Combine Human DNA and Bacteria DNA Put it into Bacteria Recombinant DNA Bacteria Makes The Protein We Want! Yay!!!!

Why use bacteria?

Box #4 Describe recombinant DNA.

Now, time for a bit more complicated example… Gives resistance to Antibiotics Can grow on Agar plates with antibiotics lacZ gene allows Bacteria to break down the Sugar lactose So, if lacZ is cut, Can the sugar Lactose, be broken Down?

Has the restriction Site we are looking for

The lacZ gene breaks Down Lactose and the Colonies turn blue When the sugar is Broken down. That’s how we know that it is NOT recombinant DNA

But there are different kinds of Recombinant plasmids…. how do we isolate Our gene of interest???

Time to check ourselves…

We have two options… Look for the gene itself or Look for the protein product

To find the gene itself…. We move the white ones because we know that the lacZ gene doesn’t work, so it must be recombinant. Some of those recombinants have our gene of interest

If we know the DNA sequence of our gene, we can find it!

Nucleic Acid Hybridization

1 st : Denature DNA 2nd: Use radioactive RNA probe to bond With the DNA 3rd: Once we know Which colonies have The probe, put them in A large liquid culture And isolate the gene

Box #5 Describe how nucleic acid hybridization helps to find the gene of interest.

Genomic Libraries All of these recombinant DNA’s can be saved And shared with other scientists

Complimentary DNA (cDNA) cDNA libraries already have the introns cut out Used to study gene expression throughout development

What happens if you only have a small amount of DNA or an impure DNA sample? Use Polymerase Chain Reaction!

Polymerase Chain Reaction (PCR)

What do we do if we want to compare Nucleotide sequences?

Box #6 When is PCR used? What does it make? What kind of special enzyme does it have?

Southern Blotting Used when too many bands to distinguish It combines Gel electrophoresis and nucleic acid hybridization

Box #7 How does southern blot combine gel electrophoresis and nucleic acid hybridization?

RFLPs Restriction Fragment Length Polymorphisms Differences in the restriction sites on homologous chromosomes that result in different restriction patterns

In gel electrophoresis, the DNA fragments that have been cut by restriction enzymes are loaded into wells in a gel made of agarose.

The agarose gel is submerged in a chamber filled with highly conductive salt solution (buffer).

DNA is a negatively charged molecule due to the phosphate groups.

When an electrical current is applied to the chamber, the fragments of DNA will migrate toward the positive electrode.

Different size fragments will move at different speeds through the gel; smaller fragments will migrate further than longer fragments in the same period of time. This separates the DNA fragments by size. Direction of Current Larger Fragments Smaller Fragments

The gel is then stained with ethidium bromide, which binds to the DNA and makes it easier to see when viewed under a UV light.

The size of the DNA fragments can be determined by comparing them to markers, DNA fragments of known size.

Box #8 Explain the steps of Gel electrophoresis and what its used for.

Box #9 Explain at least two commonly used technologies you just learned about that can be used to manipulate heritable information.

Products of genetic engineering Genetically modified foods Transgenic animals Cloned animals Pharmaceuticals, such as human insulin

Genetically modified (GM) foods

Monsanto’s Roundup Ready Products Monsanto’s Roundup Ready (glyphosate resistant) crops include corn, wheat, soybeans, canola, cotton, sugarbeets, and alfalfa. Monsanto’s Roundup Ready (glyphosate resistant) crops include corn, wheat, soybeans, canola, cotton, sugarbeets, and alfalfa.

Commercial sale of genetically modified foods began in 1994, when Calgene first marketed its Flavr Savr delayed ripening tomato.

GM foods have been engineered for faster growth, resistance to pathogens, production of extra nutrients, and many other purposes.

Transgenic animals are animals with foreign DNA incorporated into their genome.

GM livestock exists, but is currently not on the market as food.

Most transgenic animals are mice, which have been manipulated to express human diseases for medical research.

Transgenic pigs may provide organs for human transplant.

Products such as insulin, growth hormone, and blood anti-clotting factors available from the milk of transgenic cows, sheep, or goats.

Recombinant DNA technology to modify E. coli bacteria to produce human insulin was one of the earliest uses of biotechnology in pharmaceutical manufacturing.

Box #10 How can changes in a specific DNA or RNA sequence result in changes in gene expression (phenotype)?