BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

BIOTECHNOLOGY-intentional manipulation of genetic material of an

organism

•

• 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

Deoxyribonucleic Acid (DNA)

Four bases:AdenineThymineGuanineCytosine

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.

Genome: the entire sequence of DNA

Gene: the part of the code that corresponds to a protein

*genes can be transferred from one organism to another*

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

Plasmid:

- 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. 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”

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:

1. Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends.

Insert

2. Combine the sticky ends using ligase.

ligase: enzyme used to join DNA together

3. Introduce the recombinant plasmid into bacteria.

Making a Recombinant Plasmid

Bacterial Transformation

- - - - - -

- - - -- - - -

- - - - - -

- - -

+ +

+ +

+- ++ -++ - - ++ - ++

+ +

+ +

+

+ - +

phospholipid bilayer

plasmid

Ca2+ ions

• 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

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:

• the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

• where the plasmid starts to duplicate itself

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!

• In order to do these things, we need a way to make many copies of the genes we want

• easy to grow • no ethical issues• small genome • easy to

manipulate

Using Bacteria as Production Factories

Making an insert: Polymerase Chain Reaction

Common uses of biotechnology:1. Making "stuff”

• proteins, enzymes, medication, etc. can be produced by engineered bacteria!

• Food can be altered to have new traits• Cloning (therapeutic and reproductive)

2. Genetic screening• crime cases, relationship, genetic screening, etc.

3. Gene Therapy

Therapeutic cloning

• used to produce tissue that is identical to the donor, to prevent rejection

Reproductive Cloning• creates an organism with the same

genetic material (DNA) as the original organism – an EXACT COPY of the donor

Dolly the Sheep

• the first cloned sheep

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

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