Genetic Engineering • Intent of altering human genome • Introducing new genetic material into genome• Isolating genes to produce on large scale (Insulin)

Recombinant DNA• DNA that contains

genes of two species • How?

– Restriction enzymes – cut out desired gene

– Occur naturally in prokaryotic cells

– Recognize specific recognition sites – 4 to 8 base pairs

– Recognition sites are palindromes

– Cuts gene (digests) in one direction only

– Creates restriction fragments

Process – Restriction Enzyme• Locates

recognition site (Top Strand)

• Cuts the DNA backbone

• Locates recognition site (Bottom Strand)

• Cuts the DNA backbone

• DNA separates

Sticky Ends or Blunt Ends• Sticky ends – zigzag cuts in strand• Blunt ends – straight cut across strand

Putting Fragments together • DNA ligase – sticky ends • T4 DNA ligase – blunt ends – forms phosphodiester bonds in DNA

Plasmids • Small circular pieces

of DNA found in bacteria

• Used as vectors for recombinant DNA (artificial)

• Restriction enzymes used to isolate specific gene are used to cut plasmids

Process • Plasmids and DNA

fragments are placed in same solution

• Anneal • DNA ligase is used

to form phosphdiester bond

• Recombinant DNA introduced into host cell

• DNA is cloned

Identifying Bacteria Clones with Target Genes• Hybridization –

identify cells that contain recombinant DNA

• Identified using a hybridization probe – short single stranded complementary DNA molecule

• Once identified bacteria can be grown in huge quantities (commercial use)

Transformation • Cells that receive

foreign DNA• Bacterial cells

sometimes will not take up a plasmid

• Bacteria are placed in ice water bath containing CaCl₂

• Solution is heated and cooled repeatedly disrupting plasma membrane of bacteria allowing plasmid to enter

• Solution is kept at 37⁰C to stabilize and grow

Cloning Insulin

Restriction Maps• Diagrams that show all recognition sites on a

specific plasmid and distances in base pairs• Shows which restriction enzyme should be used• Allow scientists to determine which plasmids will

work the best for cloning experiments

Gel Electrophoresis• Technique used to separate fragments of

DNA (PCR)• Used to help identify information about

unknown pieces of DNA (restriction maps/forensics)

PCR• Polymerase Chain Reaction– Increase number of DNA copies from a single

biological sample in a few hours– Only specific regions of a chromosome are replicated

• Process– Denaturation– Annealing– Elongation

Taq polymerase is used to put strand together Isolated from bacteria that live in hot springs

Genetic Engineering• Biopharming – Pharmaceutical products produced on large scale – Organisms are genetically engineered to produce a

specific protein – Ability to make new protein is passed on to offspring

Genetic Engineering• Transgenic Organism (genetically modified

organism, GMO)– Organisms that contain one or more genes from another

organism

Knockout Mice

Why use Genetic Engineering?• Cost • Larger organisms

can produce larger molecules

• Better versions of organisms

• 80% of Canadas Canola crop is GM

Gene Therapy • Techniques used to replace, remove or alter a defective gene

before symptoms are expressed• Insert healthy genes into cells of tissues that are affected by a

disorder

Gene Therapy

Gene Therapy• Germ-line gene

therapy– Genes

introduced in sperm or egg cells

– Passed on to future generations

• Somatic gene therapy– Genes

introduced into body cells (not sperm or egg)

– Will not be passed on

Biotechnology• Artificial Insemination– Sperm is collected and

concentrated before being placed in woman’s uterus

• In Vitro Fertilization– Eggs from mom are

retrieved and combined with sperm in petri dish

– After fertilization, zygote is placed in woman’s uterus

Genetic Testing• Determining if your baby carries a gene that is

associated with an increased risk of developing a disease

Aminocentesis• Benefits – Diagnosis of

chromosome abnormalities

– Diagnosis of other defects

• Risks

– Discomfort for expectant mother

– Slight risk of infection– Miscarriage

Chorionic Villus Sampling • Benefits– Diagnosis of

chromosome abnormalities

– Diagnosis of certain genetic defects

• Risks

– Miscarriage– Infection– Newborn having limb

defects

Stem Cell – an undifferentiated cell that can

divide to form specialized cells

Embryonic Stem Cells – differentiate into any kind of

cell

Muscle Cell

Skin Cell

Nerve Cell

Heart Cell

Tissue Stem Cells (Adult Stem Cells) – exist within specialized tissue,

differentiate into certain types of cell s

Bone Marrow

Stem Cells

Cord Blood Banking• Source of hematopoietic

(blood forming) stem cells• Blood (stem cells) found in

umbilical cord after birth• Similar to tissue stem cells• Develop into various blood

cells• These stem cells could be

banked (stored)• Used to treat variety of

diseases

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