Manipulating Genomes


What is DNA Sequencing?

DNA sequencing determines the precise order of nucleotides within a DNA molecule.

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The principles of DNA Sequencing.

1. MIXING - DNA sample for sequencing is mixed with radioactive primers (allows visulisation on the the gel), 4 DNA nucleotides and a DNA polymerase.

2. ADDING TERMINATOR BASES - Add small amount of terminator bases, special modified dideoxy nucleotides, that cannot form phosphodiester bonds and so cannot further synthesise, for example, A* which is also radioactively labelled.

3. SYNTHESIS - Let DNA polymerase synthesise many DNA copies of the sample, using a thermal cycler like used in PCR, until randomly dideoxy nucleotide is added to the growing chain and stopping the synthesis of that chain. Results in a range of DNA molecule synthesised ranging from full length to very short.

4. VISULISATION - Contents of test tubes run side by side on electro gel and DNA is visualised by autoradiography. Since the fragments are sorted by length, they can simply read off gel starting with the smallest fragment at the bottom and working up.

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First generation DNA sequencing.

Chain Termination Method:
1. Mixture of following added to 4 separate tubes: a single-stranded DNA template, DNA polymerase, lots of DNA primer, free nucleotides and fluorescently-labelled modified dideoxy nucleotides e.g. A*.
2. Tubes undergo PCR-like cycles to produce many strands of DNA of varying lengths as each one terminates at different points depending on when terminator base was added.
3. DNA fragments in each tube are separated by capillary electrophoresis which is good for separation in a single tube.
4. Gel is visualised by laser beam (UV light) and complementary base sequence can be read from the gel, starting with the smallest nucleotide at the bottom of gel because it is able to travel the furthest. Each band after represents one more base added.

Cycle Sequencing:
Completely automated chain termination, however the sequence of colour on gel is converted to DNA sequence by a computer programme.

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Second generation DNA sequencing.

Massively Parallel Sequencing:

Rather than using gel or capillary tubes, sequencing takes place on a plastic slide known as a flow cell.

Millions of DNA fragments attached to slide and replicated in situ using PCR to form clusters of identical DNA fragments.

Clusters sequenced and imaged at the same time using the principle of coloured terminator bases to stop reaction so an image can be taken.

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Whole genome sequencing.

Bacterial artificial chromosomes (BACs):
Used for entire genome, all the DNA, of an organism.

1. Genome cut into smaller fragments using restriction enzymes.
2. Fragments inserted into BACs - man-made plasmids.
3. Each BAC inserted into individual bacterium.
4. Bacteria divide, creating colonies of cloned cells that all contain specific DNA fragment.
5. DNA extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA.
6. Each piece of DNA sequenced using the chain-termination method.
7. The pieces are put back in order to full sequence from that BAC, using powerful computer systems.
8. Finally DNA fragments from all BACs are put in order by computers to complete the entire genome.

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Gene sequencing and predicting amino acids.

Spliceosomes - pre mRNA modified to remove intros and some exon before lining up on ribosomes, exon to be translated joined by enzyme complexes called spliceosomes to give mature mRNA, resulting in single gene producing several versions of functions mRNA which code for different amino acid arrangements and so different portions and therefore several different phenotype. (Genomics)

Studying of amino acid sequences, from DNA sequencing of triplet bases predicting amino acid sequence and primary structure, highlights the complexity of relationship between phenotype and genotype - some proteins modified after synthesised and other proteins coded for may stay intact of be shortened/lengthed to give variety of proteins (proteomics).

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Gene sequencings and synthetic biology.

Synthetic biology - large field including building biological systems from artificially made molecules to see if they work in the way that we think they and redesigning biological systems to perform better and include new molecules, as well as, designing new biological systems and molecules that don't exist in natural world that could be useful to humans e.g. fuel and drugs.

Genetic engineering - single changes in biological pathways or major modification of entire organism.

Use of biological systems in industry - fixed or immobilised enzymes and drug production from microorganisms.

Synthesis of new genes to replace faulty ones - treating cystic fibrosis.

Synthesis of entire new organism - 2010 atifical genome for bacterium successfully created and replaced original genome.

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Gene sequencing and genome-wide comparisons.

DNA barcoding - identifying particular genome sections common to all species but vary between them.

Analysing human genomes - computers analyse and compare genomes of many individuals to reveal patterns of inherited DNA and diseases to which we are vulnerable.

Analysing pathogen genomes - find infection sources, identity anti-biotic bacteria, track and monitor disease outbreaks, identity genome regions that maybe useful targets for drug development and genetic markers for vaccine use.

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Computational biology and bioinformatics.

Bioinformatics - development of software and computing tools needed to organise and analyse raw biological data, including developments of algorithms, mathematical models and statistical tests.

Computational biology - using bioinformatics data to build theoretical models of biological systems used to predict outcomes in different situations.

Searching for evolutionary relationships - DNA sequences of different organisms compared so the basic rate of mutation can be calculated so can calculate how long ago species diverged from a common ancestor.

Epidemiology - study of health and disease within a population considering distribution of disease and its causes and effect by computerised comparisons of genomes of people with and without disease to detect mutations.

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What are DNA profiles and what are they used for?

An analysis of the number of times a sequence is repeated at different, specific loci in a person's genome, and so the number of nucleotides there, using electrophoresis.
Probability of two individuals having same DNA profile is very low as it is unlikely two individuals will have same number of sequence repeats at each locus in DNA.

Forensic science - DNA traces from blood, semen, saliva, hair roots or skin cells
Paternity - immigration cases to prove or disprove family relationships and demonstrate evolutionary relationships
Medical diagnosis - genetic diseases, when specific mutation is unknown or there are several mutations could have happened as identifies a broader, altered genetic pattern
Research - in vitro cloning and phylogenetic analysis

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The principles of DNA profiling.

1. EXTRACT DNA - extracting DNA from tissue sample and amplifying using PCR.

2. DIGEST DNA - digesting DNA sample by cutting into smaller fragments using restriction endonucleases that cut at different restriction sites, forming 2 cuts, one through each DNA strand to give a blunt and sticky end.

3. SEPARATION OF DNA - separating the DNA fragments using gel electrophoresis which utilises way particles move through gel medium under influence of electric current and then emerging gel into alkaline solution to separate strands which are transferred onto membrane by southern blotting.

4. HYBRIDISATION - radioactive or fluorescent DNA probes (RNA/DNA sequences) added in excess to DNA on membrane which bind to complementary DNA strands under set pH and temp, the excess probes are washed off.

5. DEVELOPMENT OF PROFILE - radioactive probes labelled with DNA fragments so that they can be used with radio graphic film or fluorescent probes are visulised with UV light and a photo is takken.

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The polymerase chain reaction.

Artificial version of the natural process by which DNA replicates that is used to produce lots of DNA from a tiny sample.
DNA sample is amplified using an excess of 4 bases in form of deoxynucleotides triphosphates, a small amount of primer DNA sequences (complementary to bases at start of fragment wanted) and enzyme DNA polymerase mixed in vial in PCR machine.

1. MIXING - reaction mixture is set up contains DNA sample, free nucleotides, primers and DNA polymerase.

2. SEPARATION - DNA strands separated by heating to 95 degrees Celsius for 30 seconds to denature the DNA by breaking the hydrogen bonds.

3. ANNEALING OF PRIMERS - temperature decreased to between 55-60 so that primers anneal to the end of DNA strands.

4. SYNTHESIS - temperature increased to 72-75 for one miniature so DNA polymerase adds bases to primer building up complementary DNA strands and producing double stranded DNA identical to the original.

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The method of electrophoresis.

Electrophoresis is a procedure that uses an electric current to separate out different sized DNA fragments, RNA fragments or proteins using a positive charge to draw DNA towards the anode, but through a porous gel such as agarose so that shorter fragments travel faster through the pores than long ones.
1. PUT IN GEL - gel poured into a gel tray and left to solidify and a row of wells created at one end, then put gel tray into a box and DNA fragments mixed with same vol of loading dye put into gel, using micropipettes, that contains buffering solution to maintain constant pH, with known length DNA put into 1st and final wells to provide reference for fragment sizing.
2. ELECTRIC CURRENT - Put lid on gel box and connect the leads to it from the power supply which is turned on to the required voltage causing the negatively charged DNA (due negative phosphate group) to move towards anode end. Let gel run for 30 mins.
3. SEPARATION - remove gel tray from box and tip off any excess buffer and place in a alkaline bugger to denature fragments so two DNA strands separate and expose bases.
4. SOUTHERN BLOTTING - strands transferred onto nylon membrane, which is covered in dry absorbent paper to drawn alkaline solution carrying DNA through by capillary action. Single DNA fragments unable to pass through and are fixed in place on membrane, at same position as on gel, using UV light or heated at 80 degrees.

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Using restriction enzymes.

A method of getting DNA fragments from an organism's DNA.
Some DNA sections have palindromic sequences of nucleotides, which consists of anti parallel base pairs.

Restriction enzymes recognise specific palindromic sequences and cut the DNA at these places.

Different restriction enzymes cut at different specific recognition sequences due the shape of the recognition sequence being complementary to an enzyme's active site.

If recognition sequences are present at either side of DNA fragment, restriction enzymes can be used to separate from the rest of the DNA.

DNA sample is incubated with specific restriction enzyme, which cuts DNA fragment by hydrolysis. The cut leaves sticky ends, small tails of unpaired bases at each end, which can be used to anneal to DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

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What is genetic engineering?

The proccess that involve extracting a specific gene from one organism, or manufacturing a gene, and placing the gene in another organism such that the receiving organism expresses the gene product.

Organism which have had genes from another species transferred do to them are described at transgenic and have recombinant DNA (consisting of DNA fragments from different organism artificially bonded together).

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The process of genetic engineering.

1. ISOLATION - isolating the desired gene using restriction endonucleases from bacteria to cut the DNA at recognition sites (8-20 base pairs long) straight across the sugar phosphosphate backbone to give blunt ends and staggered cuts to give 2 sicky ends which have a single strand of DNA which are complementary to each other so willl join together if cut by same enzyme.
2. INSERTION - inserting gene into a vector (plasmid - circular piece of DNA in bacteria), same restriction endonuclease used to cut vector so ends of vector are complementary to DNA stick ends so DNA ligase is used to glue sticky ends together forming a phosphodiester bonds between sugar and phosphate groups on the DNA backbone to form recombinant DNA with the desired gene in a process called ligation.
3. TRANSFOMATION - plasmid introduced into host cell by being mixed together with calcium on ice, the temperature of culture medium is briefly increase to 42 decrees or introduce an electrical field (electropolation) so bacteria membranes become permeable and plasmid can pass through cell membrane (or use electro fusion which fuses cell and nuclear membrane to form hybrid/polyploid cells contains DNA from both -in GM plants).
4. IDENTIFICATION - grow bacteria on medium constraining antibiotics, transformed bacteria will be able to survive (use fluorescent markers such as those from jellyfish).
5. GROWTH - cloning of the successful host cells (extrapolation).

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What is biotechnology?

The industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products by applying organisms or enzymes from organism to synthesis, breakdown or transformation of materials.

Parts of living organisms e.g. enzymes can either be contained within cells of the microorganism, intracellular, or not contained with the cell, isolated (if secreted naturally, extracellular, it is cheaper).

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Microbes are idea for use in biotechnology.

  • No welfare issues to consider
  • Enormous range of microbes capable of carrying out many different reactions
  • Can be genetically engineered to carry out process that they wouldn't normally to produce specific products
  • Short life cycle so they grow rapidly under optimum conditions
  • Single nutritional requirements that are cheap compared to chemical-based process or consume waste products which would otherwise be useless or toxic to humans
  • Easy to create ideal growth conditions which aren't expensive
  • Grow anywhere in the world at any time of year with correct conditions
  • Produce purer forms of products than in chemical processes
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The uses of microorganisms in biotechnology.

Brewing - making beer using yeast.Yeast added to type of grain and other ingredients, yeast resibres anaerobically using glucose from grand to produce ethanol and CO2.
Baking - making bread rise using yeast.Fermentation of sugars in dough to produce CO2 to ensure it doesn't stay flat.
Cheese making - using rennet contain chymosin enzymes and lactic acid bacteria. Chymosin used to clot milk is extracted from lining of calf stomach or from yeast cells that are genetically modified to produce it. Uses lactic acid bacteria to convert lactose in milk into lactic acid to turn it sour and helps to solidify.
Yoghurt production - using lactic acid bacteria.Clots and thickens milk before colours and flavours are added.
Penicillin production - formed during stress by fungi from Penicillium gene to produce antibiotic. Semi-continuous batch process with temp 25-27 degrees, buffer at about 6.5, rich nutrient medium, continuous stirring, small fermenter to maintain high oxygenation.
Insulin production - genetically modified bacteria, human insulin gene inserted.
Grown in industrial fermenter on massive scale, insulin collected and purified.
Bioremediation - use microbes to break down pollutants in soil and water. Naturally at site, extra nutrients & better growth conditions) or GM organisms that breakdown materials not normally encountered.

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Advantages of using microbes for food production.

  • Microorganisms used to make single-called protein, which can be grown using many different organic substrates including waste
  • Grown quickly, easily and cheaply
  • Cultured anywhere with right conditions
  • Single-called protection is a healthier alternative to meat
  • Do not produce positions that contaminate products
  • High yield
  • Do not need extreme conditions
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Disadvantages of using microbes in producing food.

  • Not effective if optimum conditions are not met
  • Optimum growth conditions often shared with bad microbes which can be dangerous and spoils food so need sterilisation
  • Ethical concerns about genetic manipulation
  • Unappealing if made from waste products
  • Single-called protein doesn't have same texture as real meat and can cause problems if consumed in great quantities due to high uric acid release when large amounts of amino acid are produced during breakdown
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Indirect and direct food production.

Indirect - microbes not food themselves but are upon substrate which changes its characteristics due to the bacteria's metabolic activities.

Direct - microbes consumed direct such as fungi, a good source of single-celled protein (quorn).

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What is aseptic technique and why is it used?

Aseptic techniques are measures taken to ensure asepsis - the absence of unwanted microorganisms. These could include: sterilising glasswear, minimising time agar plate is open, pass neck of broth through flame before closing and after opening lid, disinfection surface, tying hair back, working near Bunsen flame as hot air rises and draws microorganisms away.

Microbes must be able to synthesise or breakdown target chemicals but have a low mutation risk and not produce any positions that contaminate the product, so strict health and safety guidelines have to be followed when culturing non-pathogenic strains because:

1. Mutations may occur making it pathogenic

2. Pathogenic microbes may be co-cultured which makes competition for nutrients and space, lowers yield, spoils product, destroy culture and may produce toxic chemicals

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How to inncoulate a broth.

1. Make a suspension of bacteria to be grown and mix with known volume of sterile nutrients broth in a flask.
2. Stopper the flask with cotton wool to prevent contamination from the air.
3. Incubate at suitable temperature, shaking regularly to aerate providing O2 for growing bacteria.

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How to inoculate agar.

1. Wire inoculating loop is steralised by holding in a Bunsen flame until it is growing red hot - not touching any surfaces as it cools.
2. Dip steralised loop in bacterial suspension.
3. Remove Petri dish lid and make zig-zag movements across agar with loop digging into the agar by holding it horizontal.
4. Replace lid and hold down with tape with sealing completely to allow O2 to enter, preventing growth of anaerobic bacteria.
5. Incubate at suitable temperature.

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Optimum conditions when culturing a microbe.

  • pH - probe
  • temperature - water jacket surrounding vessel
  • O2 supply - pump in sterile air when needed
  • Nutrient concentration - contact between microbes and fresh medium maintained by paddles that circulate it around vessel
  • Contamination - sterilise vessel between uses with superheated steam

In bioreactors you must also:

  • Mix continuously to ensure even nutrients, O2 and temperature distribution so it doesn't become viscous
  • Aspesis so contamination by microbes from air or worked doesn't affect product yield
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The standard growth curve of a microorganism.

In a closed culture, a population of microorganisms folllow a standard growth curve:
1. Lag phase - population slowly increase as bacteria adapt to environment, start to grow and produce enzymes and other molecules, low reproduction rate.
2. Exponential (log) phase - growth is close to theoretical maximum as culture conditions are at there most favourable.
3. Stationary phase - zero net growth as death rate equals the reproductive rate, dying due to build up of poisonous waste products & lack of food.
4. Decline/death phase - death rate greater than reproductive rate as food becomes scarce and waste products approach toxic levels.

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What is batch fermentation?

Microorganisms grown in individual batches in fermentation vessel with specific nutrient solution and is removed when the culture ends (closed culture).

  • Microbes inoculated into fixed volume of medium
  • Increase in biomass follows, consuming nutrients and products
  • Stationary phase is reached when production of secondary metabolites occur
  • Process stopped before death phase and products are harvested
  • Batch equipment is cleaned and sterilised
  • Process repeated
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What is continuous fermentation?

Microorganisms continually growing inside fermentation vessel without stopping, with nutrients put in and waste products removed at a constant rate.

  • Microbes inoculated into sterile medium start to grown
  • During log phase of growth, additional sterile medium is added continuously
  • Growth conditions are adjusted to maintain optimum conditions for biomass or metabolite production
  • Primarily for sewage treatment and producing biomass

Most industrial process are semi-continuous (or batch) so a fraction of the batch is replaced with sterile fresh medium at regular intervals.

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Primary and secondary metabolites.

Primary - formed in the period of active growth as an essential part of normal functioning such as respiration producing ethanol.

Secondary - formed during stationary phases of life of culture once cell mass has reached its maximum by a non-essential process.

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What is downstream processing?

The harvested medium containing microbes, metabolites, wasted products and unused nutrients must be further processed to obtain a useful fraction.

Downward processing can be a complex process adding significant cost and time to the process.

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What is downstream processing?

The harvested medium containing microbes, metabolites, wasted products and unused nutrients must be further processed to obtain a useful fraction.

Downward processing can be a complex process adding significant cost and time to the process.

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