Biotechnology Unit

Bacterial Transformations

Plasmids used as vector for desired gene

Steps:

1. Isolate and cut desired gene out of original organism's DNA with restriction enzyme

2. Cut bacterial plasmid that has antibiotic resistance gene with same restriction enzyme used to remove desired gene.

3. Use ligase to "glue" the desired gene into bacterial plasmid

4. Bacteria takes in plasmid that has antibiotic resistance gene and is grown on agar with antibiotics.

5. you know that whichever bacterial colonies grow have taken in the plasmid with the desired gene because that same plasmid has antibiotic resistance.

6. Bacteria produce desired protein from inserted gene. (ex: human insulin)

GMOs in Agriculture

Pros

Tolerance to pests

Tolerance to herbicide

Tolerance to heat, cold, drought

Increase crop yield

Improve flavor and nutrition

Cons

Decrease in genetic diversity

Plant resistance to bacteria builds up a human resistance to antibodies

May cause intended allergic responses in consumers

Modified strains can grow in the wild and build super resistant weeds

Differs from DNA Fingerprinting

Focuses on genes (coding DNA)

Gives exact sequence of DNA rather than only size

Human Genome Project 1990-2003

Sequenced an entire human genome

Ability to compare a patient's genes with a standard genome to determine which gene(s) may be mutated, causing a disease. This allows for more effective individualized medicine and gene therapy.

Virus Vectors

Engineered to carry the desired healthy gene

Virus transported into cell within a vesicle

Virus inserts DNA with desired gene into nucleus to be transcribed and then translated into healthy protein

Ex-vivo

Cells with mutated gene are removed from body, virus inserts new gene into nucleus of cells, cells are placed back in the body.

Used to treat Severe Combined Immunodeficiency Disorder (SCID) by removing and treating blood stem cells in bone marrow.

In-vivo

Virus is administered to body and injects new gene into nucleus of cells while the cells are still inside the body.

Used to treat Cystic Fibrosis-- patients inhale the virus

Restriction Enzymes- create DNA fragments

Cut DNA at specific, usually palindromic sequences

Blunt ends: created with a straight cut (ex: Alu1)

Sticky ends: created with a staggered cut (ex: EcoR1)

Polymerase Chain Reaction: Amplify DNA fragments

Heat separates DNA strands

Taq Polymerase adds complimentary bases to each strand

Gel Electrophoresis: separates fragments by size

DNA fragments move across the gel when an electric current is applied. This is because one end of the gel is positive and one is negative. DNA has a negative charge so it is attracted to the positive charge at the other end of the gel.

Smaller DNA fragments move faster because they weigh less, so the bands furthest from the start point contain the smallest fragments.

Each person has an individual band pattern, or DNA fingerprint. This is because VNTRs (Variable Number of Tandem Repeats) in the non-coding DNA are highly variable.

Analysis of band patterns can be used for:

Crime scene investigations- matching evidence DNA with suspect DNA

Paternity tests: Each band on a child's gel must be accounted for by the father or mother. If the father's band pattern does not account for the bands on the child's gel that the mother's does not have, he is not the father.

Identifying and cataloguing endangered species.

Totipotent Stem Cells

Zygotes: can become any body or placental cell

Pluripotent Stem Cells

Embryonic Stem Cells from Blastula

Most useful type of stem cell because they can be used to create any type of tissue or organ.

Most controversial because obtaining ES kills the embryo

Mulitpotent Stem Cells

Adult Stem Cells: can become only certain types of cells

Replenish dying or damaged cells

Live in specific niches (ex: stem cells in bone marrow create all types of blood cells)

Induced Pluripotent Stem Cells

An adult cell reprogrammed to become a pluripotent cell

Has potential but has not produced results comparable to embryonic stem cells.