BIOL 111 Chapter 20

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Biotechnology and Bioethics

Definition: The use or manipulation of organisms or their components (anything in nature) to make useful products.

This practice is about 3000 years old (ancient Greek myths) — negative consequences result in negative connotations.

Are we playing God?

Will there be unexpected consequences?

Do we understand all the functions of a gene we want to change?

Is ______ something that would be acceptable in today's society?

Case Study: Neanderthal DNA Sequencing

1. Extract DNA from soft tissue (marrow) in bone samples
2. Utilize PCR to make copies of DNA
3. Use restriction enzymes to cut DNA at known locations to create fragments
4. Run DNA fragments through gel electrophoresis

Also run sequencing test to determine order of genome

Polymerase Chain Reaction (PCR)

Ingredients:

• DNA Template
• Primers based on similar DNA
• DNA Polymerase from heat-resistant bacteria
• Assorted nucleotide Bases

Process:

1. Heat up DNA solution to 90°C to separate double helix
2. Cool down slightly to 45°C to allow primers to bond to DNA templates
3. Heat back up to 72°C to allow for DNA Polymerase to make copies

Result is 2ⁿ copies of target DNA strands

Gel Electrophoresis

Ingredients:

• Gel Matrix
• Negatively charged particles
• Electrical field

Process:

1. Load particles of various sizes into wells in gel matrix
2. apply electrical field across gel, with positive anode at farthest point from wells
3. let it sit for a while

Larger fragments won't move as far as smaller fragments; result is similar to chromatography separation of particles.

Sequencing Test

Similar to PCR, only some nucleotides are fluorescently tagged:

dideoxy chain termination

DNA Pol cannot attach next nucleotide to dideoxy/tagged base

Result is a lot of fragments of different sizes; allows us to determine exact order of nucleotides:

DNA Sequence

5' TTGTCGAAGTCA 3'

Fragments (bold represents tagged nucleotides)

5' T 3'

5' T T 3'

5' TT G 3'

5' TTG T 3'

5' TTGT C 3'

5' TTGTC G 3'

5' TTGTCG A 3'

5' TTGTCGA A 3'

5' TTGTCGAA G 3'

5' TTGTCGAAG T 3'

5' TTGTCGAAGT C 3'

5' TTGTCGAAGTC A 3'

5' TTGTCGAAGTCA G 3'

Conclusion of case study

Best explanation is that Neanderthals interbred with Homo sapiens

Storage of DNA for future analysis

How do you store DNA for long-term?

Put it in a machine and let that machine store it and copy it.

DNA Cloning

Ingredients:

• Specific DNA
• Bacterial Plasmid with Selector genes
• Restriction Enzymes
• Ligase
• E. Coli Bacteria

Process:

1. Choose two Selector Genes on plasmid that is carrying fragment:
   1. amp^R - resistance to ampicillin
   2. lacZ - production of dye-reactant chemical
2. Use sticky ends from cut ends of DNA (cut by restriction enzymes) to cut same fragment on bacterial DNA (plasmids)

   This restriction site on bacterial plasmid is in the middle of one of the selector genes, so incorporation of a fragment will disrupt this gene

3. Use Ligase to glue the pieces together at known sequences on plasmids
4. Heat-shock the bacteria so they will incorporate the DNA fragment
5. Expose bacteria to ampicillin and dye: The colonies carrying fragments will be alive and not colored.

Organism Cloning

Very controversial subject...

Produce genetically identical organism from a donated "parent" nucleus/cell

Plants

Discovered in 1950s

Totipotent: one cell has potential to become a complete identical organisms

1. cut small pieces of carrot root
2. place pieces in nutrient-rich medium; shake to dissociate single cells
3. some cells began to divide
4. embryonic plant developed from cultured single cell
5. plant continued to grow in sterile environment
6. eventually produced adult plant.

Animals

Attempted in 1950s and again in 1970s

Range of potencies: cells are more likely to turn into an adult organism when they are younger

1. isolate frog embryo cell
2. put embryonic nucleus inside enucleated egg cell.
3. activate egg to begin development
4. most develop into tadpoles

1. isolate frog intestine cell
2. put intestinal nucleus inside enucleated egg cell.
3. activate egg to begin development
4. Most stopped developing before tadpole stage

More recently

1997

Dolly - sheep

1998

George and Charlie - cows

2002

CC (carbon-copy) - cat

Additional Steps

1. Culture donor cells before combining with egg (de-differentiation
2. fuse entire donor cell and enucleated egg
3. grow embryo in culture for a few days
4. implant in surrogate mother

Genetic Issues

(See BIOL 111 Chapter 15#Methylation of Alleles→)

• X-chromosome inactivation
• Differential Methylation

Why clone mammals?

Create antibodies for use in medicine

Hematech (biotech company) produces human antibodies in cattle

• Cattle have about 2.5 lbs of antibodies in their blood.
• Cattle are easy to work with and we know a lot about how they fight disease
• Technology required to inject human antibody gene and inactivate endogenous antibody genes is more advanced in cattle than in any other species.

Stem Cells

• Divide indefinitely
• Differentiate into specialized cells
• Sources: Embryo, Adult tissue via induced pluripotent (many potentials)