F215 Module 2 - Genomes and Gene Technologies

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Sequencing a Genome

The genome is broken up, and sequenced in overlapping fragments to ensure that the assembled code is accurate.

1. The genomes are mapped using microsatellites (short sequences of 3-4 base pairs) to identify their locations.

2. The samples are sheared (mechanically broken) into sections of around 100,000 base pairs.

3. Each section is put into a seperate BAC (bacterial artificial chromosomes) and transferred to E.coli cells.

To sequence a BAC section:

1. Sections are treated with different restriction enzymes, giving a number of overlapping segments.

2. Fragments are separated into size order by electrophoresis.

3. Fragments are automatically sequenced.

4. Computer programs reassemble the BAC sequence.

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How does gene sequencing allow genome-wide compari

--> Genomes can be compared to show evolutionary relationships.

--> It's possible to model the effects of possible changes to the sequnce.

--> Comparing the genomes of similar pathogenic and non-pathogenic organisms can help us to identify the genes responsible for causing the disease.

--> Analysis of an individual's genome can reveal mutant alleles, or alleles that bring increased risk for a certain disease.

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Recombinant DNA

--> Combining DNA from different organisms/sources into a single organism.

1. The required gene is obtained.

2. The gene is packaged and stabilised into a vector.

3. The vector carries the gene to the recipient cell.

4. The recipient expresses the gene through protein synthesis.

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Extracting DNA sections using restriction enzymes

--> Restriction enzymes cut DNA at a specific sequence of less than 10 base pairs (called their restriction site)

The restriction enzymes catalyse a hydrolysis of the phosphate-sugar backbone in different places, leaving a staggered cut and a few exposed, unpaired bases on each end - these are called sticky ends.

Segments cut with the same restriction enzyme have complementary sticky ends.

DNA ligase can be used to reseal the backbone.

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Electrophoresis

--> The separation of fragments of DNA in size order.

1. The (radioactive) samples are cut with restriction enzymes and placed in wells at the negative elctrode end of the electophoresis gel.

2. The gel is immersed in buffer solution and an electric current is passed through for 2 hours.

3. The DNA is negatively charged (due to its Pi groups) and so will diffuse through the gel towards the positive end. Shorter lengths of DNA move faster.

4. A nylon/nitrocellulose sheet is placed over the gell, covered with paper towels, pressed and left overnight (this is called blotting).

5. The radioactive DNA is visible is photographic film is placed over the nitrocellulose sheet.

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DNA Probes

--> Short, single stranded pieces of DNA that are complementary to the fragment that needs to be identified.

They are labelled using a radioactive marker (P-32), or a fluorescent marker.

They will anneal (bind complementarily to) the relevant section of DNA.

Probes are useful to:

- locate a gene needed for genetic engineering

- identify the same gene on various genomes (e.g for testing evolutionary relationships)

- identify the presence/absence of an allele for a particular disease

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The Polymerase Chain Reaction

Can be used to make multiple copies of DNA fragments.

1. The DNA is mixed with free nucleotides and DNA polymerase.

2. The mixture is heated to 95° - the hydrogen bonds break, and the strands separate.

3. Primers (short, single strands of 10-20 base pairs) are added.

4. The mixture is cooled to 55° and the primers anneal to either end of each DNA chain.

5. The mixture is heated to 72° (the optimum temperature for DNA polymerase - which is extracted from a bacteria found in hot springs) and DNA polymerase adds free nucleotides to the middle of the DNA chain.

PCR differs from natural DNA replication in the following ways:

  • It can only replicate short sequences.
  • It needs the addition of primer molecules 
  • A cycle of heating and cooling is needed to separate the strands, and then bind them again
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Putting DNA fragments in plasmids

1. Plasmids are cut with the same restriction enzymes as the DNA fragment was.

2. The plasmids and DNA are mixed, calcium ions are added and the mixture is heat shocked.

3. About 0.25% of the plasmids take up a DNA fragment (and are resealed with the help of DNA ligase). They are described as transgenic

Other vectors that can incorporate DNA fragments:

--> Virus genomes

--> Yeast cell chromosomes

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Conjugation

--> Bacteria can pass copies of plasmid DNA between each other - sometimes even bacteria of different species.

--> Plasmids often carry genes associated with antibiotic resistance; this swapping speeds the spread of antibiotic resistance in bacterial populations.

--> Conjugation contributes to genetic variation, helping the bacteria population to survive.

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Genetic Markers in Plasmids

Genetic markers --> genes for antibiotic resistance in plasmids. They can be used in the following procedure to identify the transgenic bacteria (the bacteria that have taken up plasmid DNA

--> Plasmids are chosen with two specific genetic markers (resistance to two antibioitics, typically ampicillin and tetracyline.

--> Plasmids are cut by a restriction enzyme in the middle of the tetracyline resistance gene (so it no longer functions).

--> The plasmids are mixed with the fragments and some are taken up by bacteria (as described on flashcard 8). The bacteria is then grown on nutrient agar.

--> Some cells are transferred to ampicillin agar - only the bacteria that have taken up a DNA plasmid will grow.

--> Some cells are transferred to tetracyline agar - only the bacteria that have taken up an 'empty' plasmid (with no DNA) will grow.

--> The bacteria that grows on ampicillin, but not on tetracyline agar is the transgenic bacteria.

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The production of insulin

1. The mRNA for the insulin-producing gene is extracted from the pancreas.

2. Reverse transcriptase synthesises a single, complementary DNA strand.

3. DNA polymerase and free nucleotides are added, and a complementary strand is synthesised. The resulting two-stranded DNA molecule (called cDNA) is an exact copy of the insulin gene.

4. Unpaired nucleotides are added to each end of the chain to form sticky ends.

5. Plasmids (from E. coli) are removed and cut with a restriction enzyme, then mixed with cDNA.

6. Some plasmids take up the cDNA to form a recombinant plasmid. They are mixed with bacteria and 0.25% of the bacteria will take up a DNA-containing fragment.

7. The transgenic bacteria are capable of producing human insulin.

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Production of Golden Rice

--> Vitamin A deficiency is a serious problem in Africa.

--> Rice plants contain the genes coding for the production of beta-carotene, but they are switched off in the endosperm (grain).

1. Phytoene synthase (from daffodils) is added, and it converts precursor molecules into phytoene.

2. Crt 1 enzyme (from bacteria) is added, and it converts phytoene to lycopene.

3. Enzymes already present in the endosperm convert lycopene to beta-carotene.

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Xenotransplantation

--> The transplantation of cell tissues or organs between animals of different species.

Pigs have been engineered to lack the enzyme alpha-1,3-transferase (a trigger for immune rejection)

There are physiological problems in the use of pig organs in humans:

  • Differences in organ size
  • A pig's lifespan is only 15 years - the organs will age quickly.
  • A pig's body temperature is 39° (2° higher than humans')

There are also ethical problems:

  • Welfare groups oppose the 'harvesting' of animals for their organs
  • Religious beliefs may prevent this type of transplantation
  • Scientists are worried about possible transfer of disease between animals
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Ethical concerns of genetic manipulation

Microorganisms

Can be engineered to produce insulin and human growth hormone

They may escape and mutate into pathogens. Antibiotic resistance is sped up due to genetic markers

Plants

Can be used to produce Golden rice, and engineered to be pest/pesticide resistant

The genes may pass to wild relatives, creating 'superweeds'. They may be toxic or cause an allergic reaction in humans/animals

Animals

Can be engineered to have more mass, produce more milk, and grow compatible organs for transplant

Animal welfare concerns and religious concerns may hinder this

Humans

Gene therapy can help cure genetic diseases

The effects are unpredictable and with germline gene therapy, the individual does not have a choice (as they are an embryo at the time of therapy)

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Somatic Gene Therapy

Augmentation

A faulty allele for a gene may lead to the organism being unable to synthesise an important protein. Augmentation is where a functioning copy of the gene is inserted, and the protein can now be synthesised.

Killing cells

Cancerous cells can be engineered to produce a particular protein (e.g cell surface antigens), causing an immune response. This may lead to a new way to treat cancer.

Somatic gene therapy treatments must be repeated regularly.

It is difficult to get the gene into the genome in a functioning state. Liposomes have been trialled to do this, as they are made from the same lipid bilayer as the cell membrane, and can therefore pass straight through.

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Germline gene therapy

A gene is engineered and inserted into an egg, sperm, or zygote.

It will be present in every cell of the new organism.

This practice means that the patient may pass the new gene onto their offspring.

It is currently illegal in humans because:

  • It could create a new disease
  • It could interfere with human evolution in an unknown way
  • It raises difficult moral, social and ethical questions, which must be answered first.
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Biotechnology

--> The industrial use of (parts of or whole) living oranisms to produce food, drugs etc.

Microorganisms are often used in biotechnology because:

  • They have short generation times
  • They can be genetically engineered to express chemicals
  • They grow well at relatively low temperatures
  • They are not climate dependent and so can be grown year-round, anywhere in the world
  • The products produced are often purer than the products that would be produced using the equivalent chemical process.
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The standard growth curve of microorganisms

1. Lag phase

The organisms are adjusting to their environment - synthesising the correct enzymes for the nutrients available, etc.

The population size remains fairly constant.

2. Exponential phase

Each individual has enough nutrients and space to reproduce

The population size doubles every generation

3. Stationary phase

Nutrients decline and (potentially toxic) waste builds up

The birth rate equals the death rate

4. Death phase

A lack of nutrients and a buildup of waste kills the organisms.

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Immobilising Enzymes

Adsorption - the enzymes bind to an adsorption agent (e.g porous carbon, glass beads)

The enzymes bind due to ionic links and hydrophobic interactions. Although reaction time is high, leakage (enzymes coming loose) can be a problem

Covalent bonding - the enzyes are covalently bonded to a support (e.g clay)

Only a small amount can be bound, but it is bound very strongly and leakage is rare.

Entrapment - The enzymes are trapped in a network (e.g gel beads, cellulose fibre network)

The rate can be reduced as the substrate must penetrate the trapping barrier

Membrane separation - The enzyme and substrate are separated by a partially permeable membrane

The substrate and product molecules are small enough to pass through the membrane, but the enzymes are not.

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Immobilising enzymes 2

Advantages:

The purification costs are low

The enzymes are immediately available for reuse.

The enzymes are protected by the immobilising matrix, and therefore more stable.

Disadvantages:

It requires a lot more setup time.

The enzymes may be less active as some of their active sites may be covered

If contamination occurs, it is very costly

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