Chapter 21

Every human has a unique comination of DNA in the chromosomes of their cells. The genome is an organism is all of the genetic material it contains - for eukaryotes that is the DNA in the nucleus and the mitochondria combined. The chromosomes are made up of millions of base pairs, but your genes which code for proteins only make up around 2% of your total DNA and are called exons. The large non coding regions of DNA that are rmeoved from mRNA before it is translated into a polypeptide chain are called introns.

Within introns, telomeres and centromeres are short sequences of DNA which are repeated many times, and this is known as satellite DNA. In a region known as a minisatellite, a sequence of 20-50 base pairs will be repeated hundreds of times and these occur at over 1000 locations on the human genome. A microsatellite is a smaller version of this and they appear in the same positions of chromosomes but the number of repeats varies between individuals, as different lengths of micro and mini satellites are inherited from both parents. Producing an image of thre patterns in the DNA of an individual is known as DNA profiling and is a technique emplyed by scientists to assist in the identification of individuals.

The process of producing a DNA profile has 5 main stages:

1. Extracting the DNA - the DNA mist be extracted from a tissue sample. A technique known as polmerase chain reaction (PCR), the tiniest fragment of DNA can be used to develop a profile.

2. Digesting the sample - the strands of DNA are cut into small fragments using special enzymes called restriction endonucleases, different ones cut DNA at a specific nucleotide sequence known as a restriction site or recognition site. All restriction endonucleases make two cuts, once throught each strand of the double helix. Restriction endonucleases give scientits the ability to cut DNA strands at defined pointas in the introns, and use a mixture of restriction enzymes that leave the satellite units intact so fragments at the end of the process include a mixture of intact mini and microsatellite regions.

3. Seperating DNA fragments - To produce a DNA profile, the cut fragments of DNA need to be seperated to froma  clear recognisable pattern, which is done using electrophoresis, a technique that utilises the way charged particles move through a gel medium under the influence of an electric current.. The gel is then immersed into alkali to seperate the double strands into single strands. The single stranded DNA fragments are then transferrred onto a membrane.

4. Hybridisation - Radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane. DNA probes are short RNA or DNA sequences complementary to a known DNA sequence. They bind to complementart strands of DNA uner particular conditions of pH and temperature. This is called hybridisation. DNA probes identify the microsatellite regiosn that are more varied than the larger minisatellite regions. The excess probes are washed off.

5. seeing the evidence - if radioactive lables were added to the DNA probs, X-ray images are taken of the membrane, or if fluorescent labels were added to the probes the membrane is placed under UV light so that the tags glow. The framgments will give a pattern of bars unique to the individual. 

DNA fragments are put into wells in agarose gel strips which also contain a buffering solution to maintain a constant pH. In one or ore wells DNA fragments of known lengths are used to provide a referance for fragment sizing. When an electric current is passed through the electrophoresis plate, the DNA fragments in the wells are the cathode end move through the gel towards the anode at the other end due to the negatively charged phosphate groups in DNA fragments. The rate of movement depends on the mass or length of the DNA fragments. The rate of movement depends on the mass or length of the DNA fragments as the gel has a mesh like structure that resists the movement of molecules. Smaller DNA fragments can move through the mesh more easily than large ones can, so over a period of time the fragments seperated, The gel is placed in an alkaline buffer solution to denature the fragments so the strands seperate, exposing the bases. These strands are then transferred to nitrocellulose paper and covered in several sheets of absorbant paper to draw the alkaine solution containing the DNA fragments through the membrane by capillart action, and the frgaments are transferred to the membrane as they are unable to transfer through it. 

DNA profiling is often used in solving crimes when only tiny amounts of DNA may be available. The PCR is a version of the natural process by which DNA is replicated and allows scientists to produce a lot of DNA from a tiny original sample. The DNA sample to be amplified, an excess of the four nucleotide bases, smaller primer DNA sequences and the enzyme DNA polymerase are mixed in a vial put into a thermla cycler. The temperature within the PCR machine is carefully controlled and changes rapidly at programmed intervals, triggering different stages of the process. The reaction can be repeated to produce billions of copies of the original sample:

Step 1  - Seperation: The temperature is increased to 90-95°C for 30 seconds which dentaures the DNA by breaking the H bonds holding the DNA strands together so they seperate.

Step 2  - Annealing of the primers: The temperature is decreased to 55-60°C and the primers bind (anneal) to the ends of the DNA strands. They are needed for the replication of the strands.

Step 3 - Synthesis of DNA: The temperature is increased again to 72-75°C for at least one minute, this is the optimum temperature for the DNA polymerase to work best. DNA polymerase adds bases to the primer, building up complementary strands of DNA and so prodcuing double stranded DNA identical to the original sequence. Taq polymerase is used as it is thermophillic.

DNA profiling has many uses, its best known being the field of forensic science. PCR and DNA profiling is performed on traces of DNA left at the crime scene. The DNA profiled can be compated to that of a sample taken from a suspect or run through a criminal database.

DNA profiling therefore is a very useful tool in providing evidence for either the guilt or innocence of a suspect. DNA profiling can also be used to prove the paternity of a child whne it is in doubt and can be used to prove or disprove family relationships and is used in immigration cases.

Identifying the species to which an organism belongs to can be organised using DNA profiling which is much more accurate than other methods, and is incresingly being used to demostrate the evolutionary relationship betweem different species. 

Another use of DNA profiling is in identifying individuals who are at risk of developing particular diseases. Certain non coding microsatellites or the repeating patterns they mkae have been found to be associated with an increased risk/incidence of particular diseases, inclduign various cancers and heart diseases. These specific gene marlers can be indentified and observed in DNA profiles.

Sequencing the genome involves a number of different processes. The DNA is chopped into fragments and each fragment is sequieced. The process involves terminator bases, modified versions of the four nucleotide bases, which stop DNA synthesis when they are included. An A terminator would stop DNA synthesis at the location that anA base would be added. The terminator bases are also given fluorescent tags. The sequencing process (capillary method) has largely been overtake by more complex methods but the basic principles remain the same:

• The DNA for sequencing is mixed with primer DNA, Taq DNA poymerase, an excess of normal nucelotides and terminator bases.
• The mixtire is placed in a thermal cycler that rapidly seperates the DNA strands, anneals them, and then at 60°C DNA polymerase starts tp build new DNA strands by adding nucleotides with the complementary base to the single strands of DNA.
• Each time a terminator base is incorportated instead of a normal nucleotide, the syntehsis of DNA is terminated as no more bases can be added. As the chain-terminating bases are present in lower amounts and are added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added. After many cycles all the possible DNA chains will have been produced with the reaction stopped at every base. 

• The DNA fragments are then seperated according to their length by capillary sequencing which works like gel electrophoresis in minute capillary tubes. the fluorescent markers on the terminator bases are used to identifyl the final base of each fragment. Lasers detect the different colours and thus the order of the sequence.
• The order of the bases in the capillary tibes shows the sequence of new, complementary DNA which has been made. This is used to build up the sequence of the original DNA strand. The data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them. Once a genome is assembled, scientsits can identify the genes or parts of the genome that code for specific characteristics or that are linked with particular diseases. 

DNA sequencing technologies have vecome faster and more automated. Instead of using a gel or capillaries the sequencing reaction takes place a plastic slide known as a flow cells where millions of fragments of DNA are attached to the slite and replicated in situ using PCR to form clsusters of identical DNA fragments. The sequencing process still uses coloured terminator bases byut as all the clusters are sequenced and imaged at the same time the technique is known as parallel sequencing. High-throughput sequencing combined with new cimputer technology means that 3 billion base pairs of the human genome can be sequenced in a matter of days

Bioinformatics is the development of the software and computing tools needed to organise and analyse raw biological data to make sense of the massive quantities of data being generated. Computational biology then used this data to build theortical models od biological systems which can be used to predict what will happen in different circumstances. It is the study of biodata and is important in the abalysis of the billions of base pairs in DNA, for working out the 3D structures of proteins and for understanding moleculr pathways like gene regulation. As whole genome sequencing becomes more automated and faster advances in this field continue.

The field of henetics tha applies DNA sequencing methods and computational biology tto analyse the structure and functions of genomes is called genomics. Computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vunerable. This has enourmous implications for the field of medicine in the future. However scientists recognise with the exception of a few rare genetic diseaes, that our genes work together with out environment to affect our physical characteristics, our physiology, and our likelihood tp develop certian diseases.

Sequencing the genomes of pathogens has become fast and relatively cheap, this enables:

• Doctors to find out the source of an infection, for example birfd flu or MRSA in hospitals.
• Doctors to identidy antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping to prevent the spread of antibiotic resistance. For example the bacteria that cause TB are hard to culture and some strains are resistant to most antibiotics. Whole genome analysis makes it easier to track the spread of transmision and plant successful treatment.
• Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics, such as the ebola virus.
• Scientists to identify regions in the genome of pathogens that may be useful targets in the developent of new drugs and to identify genetic markers for use in vaccines.

Using traditional methods of observation, it can be difficult to determine which species an organism belongs to or if a new species has been discovered. Genome anaylysis provides a new aid in species identification by producing stock sequences for all the different species. One useful technique is to identify particular sections of the genome that are common to alls pecies but vary between them, so comparisions can be made, this is referred to as DNA barcoding. Scientists can identify species using relatively short sections of DNA from a conserved region of the genome. For animals the region chosen in a 648 base pair section of the mitochondrial DNA in the gene cytochrome c oxidase. This section is small enough to be sequenced quickly and cheaply yet varies enough to give clear differences between species. In land plants that region of DNA does not evolve quickly enough to show differences between species, but two regions in the DNA of the chloroplsts have been identified that can be used to identify species.The barcoding system is not perfect as so far scientists have not come up with suitable regions for bacteria and fungi and may not be able to do so, but DNA sequencing has still had a big impact on classification.

Genome sequencing has also given scientists a powerful tool to help them understand the evolutionary relationships between organisms as DNA sequences of different organisms can be compared and due to the basic mutation rate of DNA how long ago two species diverged from a common ancestor can be calculated so accurate phylogenetic trees can be built.

Proteonomics is the study of the amino acid sequencing of an organism's entire protein complement. We now know that there are 20-25000 coding genes in the human DNA but a very diffeent number of unique proteins (17-18000) so there is still work to be done. More evidence is emerging that highlights the complexitiy of the relatioshi[ between the genotype and the phenotype of an individual. The DNA sequence of the genome should enable you to predict the sequence of the amino acids in all of the proteins it produces, but the evidnece is that the sequence of amino acids is not always what would be predicted from the genome sequence alone as some genes code for many different proteins.

The mRNA transcribed from the DNA in the nucleus includes both the exons and the introns. Before it lines up on the ribosomes to be translated this 'pre-mRNA' is modified. The introns are removed and in some cases some of the exons are removed as well. Then the exons to be translated are joined together by enzyme complexeds known as spliceosomes to give the mature functional mRNA. The spliceosomes may join the same exons in a variety of ways. As a result a single gene may produce several versions of mature mRNA, which  would code for different arrangements of amino acids, giving different proteins, resulting in several different phenotypes. Some proteins are modified by other proteins after synthesis. A protein that is coded for by a gene may remain intact or it may be shortened or lenghtened to give a variety of other proteins.

The ability to sequence the genome of organisms and understand how each sequence is translated into amino acids along with the ever increasing ability of computers to store, manipulate and analyse the data has lead to the development of the field known as synthetic biology which can be defined as the design and construction of novel artifical biological pathways, organisms or devices, or the redesign of biological systems. It includes techniques such as:

• Genetic engineering - this may involve a single change in a biological pathway or relatively major genetic modification of an entire organism.
• Use of biological systems or parts of biological systsems in industrial contexts, fo example the use of immobilised enzymes and the production of drugs from microorganisms.
• The syntheisis of new genes to replace faulty ones, for example in treating CF scientisits have attempted to synthesise functional genes and use them to replace faulty genes in the cells.
• The synthesis of an entire new organisms. Scientists have created an artifical genome for a bacterium and successfully replaced the original genome with a new functioning one.

Scientists have developed new nucleotide bases which can be incorporated into DNA by special enzymes and the bases fit together well, and they have introduced a small section of these into bacterium successfully - potential for synthetically expanding the genetic code.

Advances in proteonomics and molecule biotechnology mean that it is now possible to manipulate an organsism's genome to achieve a desired outcome. The basic principles of genetic engineering involve isolating a gene for a desirable characteristic in one organism and placing it into another organism using a suitable vector. The two organisms between which the genes are transferred may be the same or similar, or very different specoes. An organism that carries a gene from another organism is termed transgenic.

The first stage of successful genetic modification is to isolate the desirable gene. The most common technique uses enzymes called restriction endonnucleases to cut the required gene from the DNA of an organism. Each type of endonuclease is restricted to breaking the DNA strands at specific base sequences within the molecule. Some make a clean blunt ended cut, however many cut the two DNA strands unevenly leaving one of the strands a the DNA fragment a few bases longer than the other. These regions with unpaired exposed bases are called sticky ends and thet make it much easier to insert the desired gene in the DNA of a different organism. Another technique involves isolating the mRNA for the desired gene and using the enzyme reverse transcriptase to produce a single strand of complementary DNA. The advantage of this technique is that it makes it easier to identify the desired gene, as a particular cell will make some very specific types of mRNA.

The DNA isolated by restriction endonucleases must be inserted in a vector that can carry it into a host cell. The most commonly used vectos in genetic engineering are bacterial plasmids which are small circular molecules of DNA seperate from the chromosomal DNA that can replicate independantly. Once a plasmid gets into a new host cell it can combine with the host DNA to form what is called recombinant DNA. Plasmids are particularly effective in the formation of genetically engineered bacteria used, for example to make human proteins. The plasmids that are used as vectors are often chosen because they contain what is known as a marker gene. For example they may have been engineered to have a gene for antiobiotic resistance. This gene enables scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in media containing the antibiotic.  

To insert a DNA fragment into a plasmid, first it must be cut up. The same restriction enonuclease used to isolate the DNA fragment is used to cut the plasmid, resulting in the plasmid having complementarty sticky ends to the sticky ends of the DNA fragment. Once the complementary bases of the two sticky ends are lined up the enzyme DNA ligase forms phosphodiester bonds between the sugar and the phosphae groups on the two strands of DNA joining them together. 

The plasmids used as vectors are usually given a second marker gene which is used to show that the plasmid contains the recombinant gene. The marker will be placed in the plasmid by genetic engineering.  The plasmid is then cut by a restriction enzyme within this marker gene to insert the desired gene. If the DNA fragment is inserted successfully the marker gene will not function. Genes that produce fluorescence or an enzyme that causes a colour change in a particualr medium are more widely used as marker genes due to concerns about creating antiobiotic resistant bacteria. If a bacterium does not fluoresce or change colour, it has been engineered successfully. 

The plamid with the recombinant DNA must be transferred into the host cell in a process called transformation. One method is culture the bacterial cells in a calcium rich solution and increase the temperature, causing the bacterial membrane to become permeable and the plasmids can enter. Another method of transformation is electroporation. A small electrical current is applied to the bacteria which makes the membranes very poroous so the plasmids move into the cell. Electroporation can also be used to get DNA fragments directly into eukaryotic cells. The nw DNA will pass through the cell and nuclear membrane to fuse with the nuclear DNA. Although this technique is effective the power of the electric current has to be carefully controlled or the membrane or whole cell could be damaged or destroyed.

Another way of producing genetically modified cells is electrofusion, in which tiny electrical currents are applied to the membranes of two different cells. This fuses the cell and nuclear membranes of two different cells to form a hybrid polypoid cell containing DNA from both. It is successfully used to produce GM plants.

Electrofusion is used different in animal cells which do not fuse as easily and effectively as plant cells. Their membranes have different properties and polypoid mammal cells, do not usually survive in the body of a living organism. However electrofusion in important in the production of monoclonal antibiodies. A monoclonal antibody is produced by a combination of a cell producing one type of anitbody with a tumour cell, meaning it divides rapidly in culture. Monoclonal antibodies are used to identify pathogens in both animals and plants, and in the treatment of a number of diseases including some forms of cancer.

The techniques of genetic engineering vary between different types of organisms but the principles are the same. It is much easier to carry out genetic modification on prokaryotes than eukaryotes, and plants are easier to modify than animals. Bacteria and other microorganisms have been genetically modified to produce many different substances that are useful to people, including hormones like insulin, clotting factors for haemophiliacs, antibiotics, pure vacxines and enzymes.

One method of genetically modifying planys uses Agrobacterium tumefaciens which is bacterium which causes tumours in healthy plants. A desired gene, for example for pesticide production, herbicide resistance or higher yield is placed in the Ti plasmid of this bacterium along with a marker gene. This is then carrued directly into the plant cell DNA. The transgenic plant cells form a callus, each of which can be grown into a new transgenic plant. Transgenic plants can alsp be produced using electrofusion making polypoid cells once removing the plant cell wall, fusion, and then using plant hormones to stimulate the growth of a new plany cell, followed by callus formation.

It is much harder to engineer the DNA of animals, especially mammals, than it is to modify bacteria or plants. This is because animal cell membranes are less easy to manipulate than plant cell membranes. However it is an important technique both to enable animals to produce medically important proteins and to try and cure human genetic diseases.

Microorganisms have been modified to produce a variety of subtances in large quantities. GM microorganisms are also used to store a living record of DNA of other organisms in DNA libraries, where DNA fragments from one organism are propagated in microorganisms through genetic engineering. GM microorganisms are a widely used tool in research for developing novel medical treatments and industrial processes. Genetically engineered pathogens however are not widely used due to health and safety concerms and the modification of genomes to make pathogens more virulent or resistant for use in biological warefare is largely prohibited. Initially some were uncomfortable with inserting human genomes into microorganismis but the the results have been overwhelmingly beneficial.

All scientists have the responsibility to consider the moral and social values or ethics of their work, which are important for huma safety, animal welfare and the protection of the environment. Ethical lapses can not only casue harm but also damage the public's trust in scientists and their research which can have significant implications on the advancment of research and understanding.

Soya beans are a major world crop, half of the 250 million tonnes produced come from GM strains. In one such modification scientists have inserted a gene into soya beans so that they produce the Bt protein which toxic to many pest species that attack the plant. This strain has also been modified to be resistant to common weedkiller, so farmers can spray it to remove weeds so that all the resources of water, light and minerals are available to the beans and they do not need to use pesticides. This results in a higher yield of soya beans with less labour and less expense.  Some of the potential risks and possible problems of GM plants include:

• Pest resistance - pest-resistant cop varietoes reduce the amount of pesticide spraying, protecting the environment and helping poor farmers. However non prest insect eating predators may be damaged by the toxins in GM plants, or the pests may become resitant to the pesticides in GM crops.
• Disease resistance - Crop varieties resistant to common plant dieases can be produced, reducing crop losses/increasing yield. However transferred genes might spread to wild populations and cause problems such as superweeds.
• Herbicide resistance - Herbicides can be used to reduce competing weeds, increasing yields, but biodiversity would be reduced if herbicides are overused to destroy weeds - superweeds.

• Extended shelf life - the extended shelf life of some GM crops reduces food waste. However this may reduce the commercial value and demand for crop.
• Growing conditions - crops can grow in a wider range of conditions and survive adverse conditions, for example crop or flood resistant crops like GM rice, and this has litte disadvantage.
• Nutritional value - nutritional value of crops can be increased, for example crops can have enhanced levels of vitamins. However people may be allergic to the different proteins made in GM crops.
• Medical uses - plants could be used to produce human medicines and vaccines, and this has no disadvantages.

One of the major concerns about GM ctops is that people in LEDCs will be prevented from using them due to developers patenting their work, as the people most in need for eg drought resistant crops will be unable to affors the GM seed. Some organisisations work to develop engineered crops such as rice to support farmers in LEDCs with whom they share the technological developments without patent constraints on seed harvesting.

It is much harder to produce GM vertebrates, especially mammals, but scientists are researching the use of microinjections, which are tiny particles of gold covered in DNA and modified viruses to carry new genes into animal DNA. Such techniques are used with the aims to transfer disease resistance from animal to another, or to modify physiology in farmed animals, for example:

• Swine fever resistant pigs - scientists have successfully inserted a gene from wild African pigs into early embryos of a European pig strain guving them immunity to otherwise a fatal flu.
• Faster growing salmon- GM salmon have recieved genes from faster growing Chinook salmon, these genes cause them to produce growth hormones all year round and so they grow to full adult size in half the time of conventional salmon, making them a very efficient food source.

One of the biggest uses of genetic engineering in animals is the production of human medicines which is known as pharming. There are two aspects to this field of gene technology:

• Creating animall models - the addition or removal of genes so that animals develop certain diseases, acting as models for the developemnt of new therapies, for example knockout mice have genes deleted so that they are more likely to develop cancer.

• Creating human proteins - the introduction of human gene coding for a medically required protein. Animals are sometimes used because bacteria cannot ptoduce all of the complex proteins made by eukaryotic cells. The human genes can be introduced into the genetic material of a fertilised cow, sheep or goat egg, along with a promoter sequence so the gene is expressed only in the mammary glands. The fertilised transgenic female embryo is then returned to the mother and when the transgenic animal is born and matures, the milk it produces will contain the desired protein and can be harvested.

The processed of animal genetic engineering raise ethical concerns including whether we should use animals to act as models for human disease, does genetically modifying animals reduce them to commodoties, and is welfare compromised during the production of genetically engineered animals. 

Some human diseases such as haemophilia and SCIDS are the result of faulty mutant genes. Scientists are looking at different ways of replacing the faulty allele with a healthy one. They can remove the desired allele from healthy cells or synthesis healthy alleles in the labatory. 

Somatic gene therapy involves replacing the mutant allele with a healthy allele in the affected somatic cells. The potential for helping people with a wide range of disease is enormous including successful treatment of diseases including retinal diseasesm immune diseases, leukaemias and haemophilia. However somatic cell gene therapy is only a temporary solution for the treated individual as the healthy allele will be passed on every time a cell divides by mitosis but somatic cells have a limted life and are replaced with stem cells which will have the faulty allele, and a treated individual will still pass the faulty allele on to any children they have.

The alternative is to insert a healthy allel into germ cells, usually the eggs, or the embryo directly after fertilisation as part of IVF and the individual will be born with the normal allele in placd and would pass it on to their offspring. This called germ line gene therapy. Such therapy has been successfully done with animal embryos but it is illegal for human embryos as a result of various ethical issues including the fact that the potential impact on an individual is unknown as it is done without consent and once done the process is irrevocable.