BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

Deoxyribose Nucleic Acid (DNA) determines the characteristics of all living organisms. occurs in most cells of all organisms composed of four different nucleotides in different combinations each cell in the human body contains more than 3 BILLION letters

Four bases: Adenine Thymine Guanine Cytosine 2 bonds 3 bonds Sugar and phosphate backbone Double helix structure (two spirals around each other)

  The only difference between living organisms is the amount and order of the four nucleotide bases.

Why would we want to do this? BIOTECHNOLOGY The intentional manipulation of genetic material of an organism Why would we want to do this? To study cellular processes of an organism E.g. Glowing gene from jellyfish to tobacco plant To give one organism the trait(s) of another E.g. Anti-freeze from fish blood into strawberries to survive through early frosts

Part 1: Manipulating Bacteria: The Making of a Plasmid

bacteria exchange these plasmids to share DNA - a small circular piece of extra-chromosomal bacterial DNA, able to replicate bacteria exchange these plasmids to share DNA E.g. antibiotic resistance genes

Since plasmid is made of DNA it can code for genes, ex Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA Specific DNA sequences can be recognized by enzymes called restriction endonucleases

Restriction Endonucleases/Restriction Enzymes enzymes that are able to cut double-stranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’

Restriction enzymes can create “sticky ends or “blunt ends”

fragment end of a DNA molecule with a short single-stranded overhang Sticky Ends fragment end of a DNA molecule with a short single-stranded overhang Blunt Ends fragment end of a DNA molecule with no overhang Once made, the ends can be re-joined together by other enzymes ("enzyme glue")

To Make a Recombinant Plasmid: Insert Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. Combine the sticky ends using ligase. ligase: enzyme used to join DNA together So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA? 3. Introduce the recombinant plasmid into bacteria.

Making a Recombinant Plasmid So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA?

So, let’s REVIEW…

Bacterial Transformation introduction of foreign DNA into a bacterial cell plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell - - - - - - - - - - - - - + + + + + - ++ -++ - - ++ - ++ + - + phospholipid bilayer Ca2+ ions plasmid

Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

Example plasmid: Origin of Replication: where the plasmid starts to duplicate itself the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

Part 2: Where do we get our insert sequence? From someone else’s DNA ex. fish gene in strawberries, jellyfish gene in plants Make it! GFP, tobacco plant SO WE CAN HARVEST OUR THE DESIRED GENE, BUT WE ARE PROBABLY GOING TO NEED WAY MORE OF IT THAN IS FEASIBLE TO COLLECT - SO WE AMPLIFY IT!

Use bacterial “factories” In order to do these things, we need a way to make many copies of the genes we want Use bacterial “factories” easy to grow lots no ethical issues small genome is easy to manipulate

Using Bacteria as Production Factories easy to grow no ethical issues small genome easy to manipulate

Step 3: Taq (heat resistant) polymerase extends sequence Step 1: DNA becomes single stranded due to heat Step 2: Primers anneal (attach) to desired start sites through complementarity Step 3: Taq (heat resistant) polymerase extends sequence

Polymerase Chain Reaction Making an insert: Polymerase Chain Reaction

Common uses of biotechnology: Making "stuff” proteins, enzymes, medication, etc. can be produced by engineered bacteria! Genetic screening crime cases, relationship, genetic screening, etc. 3. Gene Therapy

Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

Gene therapy desired gene is inserted into cell's nucleus using a retrovirus as a carrier defective gene replaced by functional gene

adenosine deaminase deficiency Ex. ADA deficiency adenosine deaminase deficiency little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system