DNA Technology

The process of manipulating genes for practical purposes. Genetic Engineering The process of manipulating genes for practical purposes. Because all DNA has the same basic structure, we can cut and paste genes from one organism into the chromosome of another organism.

Recombinant DNA Vector Gene of Interest DNA made from two or more different organisms (Connecting DNA from different sources) Gene of Interest The gene to be inserted (Codes for the trait we want expressed) Vector The DNA into which we will insert the gene of interest. *Plasmids make great vectors*

Small, self-replicating circles of DNA (Found commonly in bacteria) Plasmids Small, self-replicating circles of DNA (Found commonly in bacteria) Readily take up new DNA. Self-replicating Easily transferable from one cell to another. Let’s swap DNA!

Steps in Genetic Engineering Step 1: Cut Gene of Interest & Vector with restriction enzymes. gene from other organism transformed bacteria recombinant plasmid vector plasmid cut DNA + glue DNA

How Do We Cut DNA? Restriction Enzymes Enzymes that cut DNA in specific locations “Blunt” ends CANNOT be glued to another piece of DNA. “Sticky” ends CAN be glued to another piece of DNA.

Why do we need “Sticky” ends? Sticky ends make it possible to “glue cut ends together. MUST cut Gene of interest & Vector plasmid with the SAME restriction enzyme!! MUST have Complimentary Sticky ends!

How do we “Glue” sticky ends together? Step 2: Connect DNA fragments together How do we “Glue” sticky ends together? DNA Ligase An enzyme used to connect sticky ends of DNA fragments

+ How do we get the recombinant plasmid into a cell? I feel totally transformed! Look What I found! Bacteria will pick up foreign DNA and make it their own.

How do we find the bacteria that have been transformed? We engineer a plasmid that helps us find the bacteria that pick it up: Insert the gene of interest in the middle of the LacZ gene thus destroying it. No LacZ gene = No blue pigment LacZ gene Codes for a blue protein Now culture the bacteria in a petri dish with ampicillin in it. Antibiotic resistance genes

Which cells Have the Recombinant Plasmid? Gene of interest Original plasmid Plasmid & Gene anneal 2 Plasmids anneal with one another Gene of Interest anneals with itself Die Grow (Blue) Grow (White) Culture the bacteria in agar containing ampicillin

Screening for Bacteria that Contain the Recombinant Plasmid Bacteria take up plasmid Functional LacZ gene Bacteria make blue color Bacteria take up recombinant plasmid Non-functional LacZ gene Bacteria stay white color Which colonies do we want?

How do we get more of these transformed bacteria? Single Transformed Bacteria Feed them and they will reproduce! Binary Fission Copy DNA and split = “Cloning” Culture Bacteria Harvest (purify) protein Gene Cloning

Word Processing Cut, Paste, Copy, Find… So, Transformation is like: Word Processing Cut, Paste, Copy, Find… Cut Restriction enzymes Paste Ligase Copy Plasmid replication Binary Fission Bacterial Transformation Change the meaning of the message. 14

Many Uses of Restriction Enzymes… Now that we can cut DNA… We can COMPARE it! Why? Forensics Medical diagnostics Paternity Evolutionary relationships and more… 15

Can we add those little marshmallows? Comparing DNA Cut all DNA with same restriction enzyme * Restriction fragments Separate fragments by size Gel Electrophoresis Process that uses an electrical current to separate DNA fragments by size DNA jello?? Can we add those little marshmallows? 16

“Swimming through Jello” Gel Electrophoresis A method of separating DNA fragments by size using an electrical field. DNA is negatively charged so in an electrical field it moves toward the positive side Small pieces travel faster/farther than larger pieces. DNA         – + “Swimming through Jello” 17

Preparing a Gel

DNA & Restriction enzyme Restriction Fragments Gel Electrophoresis: DNA & Restriction enzyme Restriction Fragments negative - Completed gel wells Longer fragments power source gel Shorter fragments Who’s going to win the race? + Electrical current positive 19

DNA Fingerprint A pattern of dark bands on photographic film made when an individual’s DNA restriction fragments are separated by gel electrophoresis, probed, and then exposed to an X-ray film. Because each individual’s DNA is unique, each person has a unique DNA fingerprint

DNA Fingerprint Uses: Forensics Comparing DNA sample from crime scene with suspects & victim Suspects You’re Under Arrest! Crime scene sample S1 S2 S3 V – DNA  + 21

DNA Fingerprint Uses: Paternity Cases Who’s the father? – Mom F1 F2 child DNA  + 22

PCR Process used to repeatedly copy the same piece of DNA many times. Helpful in magnifying DNA from: Crime scenes Fossil remains PCR Animation

Human Genome Project 3.2 billion base pairs in the human genome Only 1-1.5% codes for proteins Only about 30,000 to 40,000 genes

Why would we want to do this? Genetic Engineering Why would we want to do this? To Create Drugs & Vaccines: Create organisms (Animals or bacteria) that produce human proteins Sheep that secrete a human blood protein in their milk that can be used to slow lung damage in cystic fibrosis patients. Bacteria that produce: Factor VIII-protein that promotes blood clotting to treat Hemophilia Growth hormones Production of human insulin by bacteria To Grow Organs for transplantation: Mice that grow a human ear which can be used for transplant

To create better agricultural products: Genetic Engineering Why would we want to do this? To create better agricultural products: Delayed ripening & resistance to spoilage Anti-softening tomatoes Protect crops from insects: BT corn Corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) Herbicide resistance: Cotton Bacterial gene resistant to weed-killing herbicides Improve quality of food: Golden rice Rice producing vitamin A improves nutritional value

Animal Cloning Dolly 1st Cloned Animal 1996

Problems with Cloning Few survive Fatally oversized Problems in development Why does it Fail? During early development, many genes are permanently turned off by the addition of methyl groups.