Year 2- Chapter 11-manipulating Genomes Flashcards

Question

What can a multiplex PCR do

Answer 1

It involves simultaneous amplifications of numerous DNA sequences in a single reaction mixture by using more than one pair of primers.

Answer 2

Substances, associated with the stages of extracting and purifying the DNA hinder PCR reactions. This includes ionic detergents, gel loading dyes and the enzyme proteinase K, used in extracting DNA from cellular material which, if left in the mixture, will break down polymerases. Similarly, certain substances present in blood can inhibit PCR, such as haemoglobin and the anti-clotting agent heparin.

Answer 3

This is a technique for separating and identifying substances in a similar way to paper and thin-layer chromatography. Is used to analyse proteins and DNA and is carried out often on a paper or more often with gels. This involves placing a mixture of molecules into wells cut into a gel, adding a suitable buffer solution and applying an electric field.

Answer 4

Composition of the gel:polyacrylamide gel are needed for proteins and agarose for DNA Net charge of the molecule: negatively charged molecules move towards the anode (+) and positively charged molecules move towards the cathode (-); molecules with a higher charge move faster than those with less overall charge. Size: smaller protein molecules and fragments of DNA move through the ‘holes’ in the gel faster than larger ones.

Answer 5

The charge on proteins is dependent on the ionisation of the R groups on the amino acids residues. Some amino acids have R groups that are positively charged (-NH3+) whereas some of the amino acids are negatively charged (-COO-) or not charged at all.

Answer 6

Whether these R groups are charged or not depends on the pH of the solution. When proteins are separated by electrophoresis the procedure is carried out at a constant pH by using a buffer solution. Usually the proteins are denatured in a reducing agent (mercaptoethanol) that breaks disulphide bonds. The proteins are often added to sodium dodecyl sulfate (SDS) which converts these proteins to negatively charged rod-shapes that move through the gel. The smaller proteins move faster through the gel and therefore travel further than larger proteins towards the anode.

Answer 7

It can be used to separate the polypeptides produced by different genes; also used to separate the variant forms of enzymes produced by different alleles of the same gene

Answer 8

The beta globin in haemoglobin molecules normally has the amino acid called glutamic acid- this has a non-polar R group so it is uncharged. In SCA, glutamic acid is replaced by valine acid, which has a R group that is charged. The two variants of beta globin can be separated by gel electrophoresis due to their difference in net charge. Haemoglobin in SCA, have a lower negative charge than normal haemoglobin. Due to this, they do not move as far through the gel compared to normal haemoglobin.

Answer 9

All DNA fragments carry a small charge due to the negatively charged phosphate groups in the sugar-phosphate backbone of each polynucleotide.

Answer 10

The DNA fragments move through the gel towards the anode. The distance travelled by a length of DNA depends on its size- the shorter fragments travelled further. The distance that the DNA fragments travel through the gel are determined by visualising the DNA with stains such as ethidium bromide and Azure A. The tracking dye moves through the gel slightly in front of the smallest DNA fragments so that the progess of electrophoresis can be followed and stopped before reaching the end of the gel.

Answer 11

1. Melt agarose gel in buffer solution 2. Insert a toothed comb at one end of the tank to make the wells to take DNA samples 3. Pour in the agarose gel. 4. Let the gel to set. Place electrodes at either end of the tank. 5. When the gel is set, pour in buffer solution and remove the comb. 6. Add blue dye to each DNA sample 7. Add DNA and dye mixture to the wells 8. Connect electrodes to the power supply. 9. When the blue dye in within 10 mm of the end of the gel, disconnect the power supply. 10. Pour away the buffer and add DNA stain (Azure A) for 4 minutes. 11. Rinse with water and analyse the fragments of the DNA, which will appear blue.

Answer 12

In this, the fragments of DNA (marked with fluorescent markers), are detected by a laser and a sensor that detects the fluorescence as each band passes towards the anode.

Answer 13

To find the order of the nucleotide bases along regions of DNA.

Answer 14

Similar to PCR, when a newly synthesised DNA polynucleotide is being formed, free floating nucleotides are being arranged and bonded opposite to their complimentary bases. We do not know what base sequence of the polypeptide chain. So, in sanger sequencing, along with simple free-floating nucleotides, there are also nucleotides which have been marked with a fluorescent marker. These nucleotides terminate the synthesis of the DNA polynucleotide as soon as they are bonded to the rest of the nucleotide chain. Since, they have no particular order in which they bond with the polynucleotide fragment, these nucleotides stop the synthesis of the fragments at any point (in other words, they bond to the rest of the nucleotide chain randomly; at any point). This causes the fragments to be different sized. Different sized fragments are formed for every single of the four bases- so there are fluorescent terminating adenine nucleotides, thymine nucleotides, guanine nucleotides and cytosine nucleotides. Each base has been marked a different colour. The fragments can only contain one fluorescent nucleotide. The fragments are divided based on the type of terminating base they contain and poured into their base-labelled wells in the electrophoresis. During the process, the smaller fragments are able to move further and faster towards the anode, thus separating from the longer fragments. So, the fragments are ordered based on size. Each fragment will give off a colour which will indicate the base. So, while going through the size order, we can work out what bases each colour represents; thus the base sequence.

Answer 15

Each individual fragment of the DNA is read separately. Then, the overlaps are identified between the fragments. After this, the whole DNA sequence is put together.

Answer 16

Next-generation sequencing. These enable thousands of DNA molecules to be sequenced at the same time without the need for electrophoresis. They are much faster than the traditional methods such as shotgun method and sanger method. Thus, the expenses in this process are reduced.

Answer 17

When DNA flows through the upper protein, a current is produced- since DNA is formed by ions; when there is a flow of ions a current is created. Each base (A,T,C and G) alters current flow in a different way Lower protein forms a pore through lipid bilayer The change in current as each base passes through the pore is detected so the base sequence is determined.

Answer 18

Comparisons of whole genomes allow scientists to find the location of the same or similar genes in different species and also provide data to analyse evolutionary relationships between species.

Answer 19

By understanding the genome, researchers have been able to ‘knock out’ the genes in a simple organism to find out the minimum number required to keep the organism alive.

Answer 20

This has given to opportunities to design and make new molecules by writing completely new base sequences and inserting them into bacterial or eukaryotic cell using the techniques of genetic engineering. This is possible because they can use the genetic code to predict the amino acid sequence encoded by any length of DNA. This also enables t predict what shape the tertiary structure of a protein have.

Answer 21

Possible to involve enzymes in medicinal drug production Changing the sequence of bases in existing genes to improve the way in which certain proteins work; such as various forms of insulins are available to treat diabetes Redesign cellular systems so that organisms such as the genetically modified for the production of proteins, can work more efficiently.

Answer 22

This is the term coined for all the various applications of molecular and cellular biology; much of which relies on a knowledge of the structure and function of nucleic acids.

Answer 23

This is the study of the spread of diseases in populations.

Answer 24

When a nucleotide sequence is repeated in a DNA molecule, it is often considered a gene; with the variable number of repeats being considered as alleles.

Answer 25

Sequencing machines containing 96 sets of capillary flow electrophoresis apparatus. These machines increased the speed at which different lengths of DNA synthesised by chain termination could be sequenced.

Answer 26

The discovery of sequences that are repeated in DNA in structural genes and in regions between them led to methods of genetic profiling.

Answer 27

Among first markers to be used were minisatellites, which are sequences repeated at various points along chromosomes. They can cut by restriction enzymes and the lengths of the fragments determined by gel electrophoresis.

Answer 28

STRs, short tandem repeats, otherwise known as microsatellites. These are repeated sequences of nucleotides which are much shorter than minisatellites. They are regions of the genome composed of 2-5 nucleotides repeated 10-30 times. The number of repeats in any one STR is variable. This variability is caused by the inaccuracy of DNA polymerase in copying these regions during replication. The error rate in replication that leads to mutation is very low, but one effect is an increase in the number of these repeated base pairs. STR markers can be simple (identical length repeated sequences); compound (two or more adjacent repeated sequences) or complex (multiple length repeated sequences).

Answer 29

The STRs show a high degree of polymorphism, making them of particular use to the forensic scientist, and the STR regions are in non-coding DNA so there is no selective pressure against any of the alleles, the result that there is much variation between different people. They tend to be four-five nucleotide repeats, as they are robust, suffer only low levels of environmental degradation and provide a high degree of error-free data. They represent discrete alleles that are distinguishable from one another, they show a great power of discrimination, only a small amount of sample is required due to short length of STRs.

Answer 30

PCR is used to make copies of the DNA. Then, the sequences are separated by gel electrophoresis. The STRs are labelled with fluorescent dyes and detected by a laser scanner. More modern analysis dispenses the electrophoresis stage and detects the fluorescence during PCR with four colours (e.g red, green, blue and yellow). A print out will form displqying the peaks.

Answer 31

Combining biological data with computer technology and statistics. It builds up databases and allows links to be made between them. The database holds gene sequences, sequences of complete genomes, amino acid sequences of proteins and details of protein structures. Databases can also hold data on macromolecules: their structures and functions, gene expression patterns, metabolic pathways and control cascades. A future problem is collecting and organising data on the variety of proteins that are synthesised by eukaryotic cells.

Answer 32

The Genome OnLine Database (GOLD) is a comprehensive online resource to catalogue and monitor genetic studies worldwide. It provides up-to-date status on complete and ongoing sequencing projects along with a broad array of curated metadata. Nucleotide sequences are held by the Nucleotide Sequence Collaboration between GenBank (USA), the European Nucleotide Archive and the Center for information Biology and DNA Data Bank in Japan. These databases compare sequences between the different organisations on a daily basis. The database Ensemble holds data on the genomes of eukaryotic organisms. Among others it holds the human genome and the genomes of zebrafish and mice that are model organisms used a great deal in research.

Answer 33

Research and analysis Retrieval(search) tool, BLAST, is an algorithm for comparing primary biological sequence information, such as the primary sequences of different proteins or the nucleotide sequences of genes. Use to find similarities between sequences that they are studying Sequences can be matched degrees of similarity can be calculated after a whole genome is sequenced. Very close similarity indicate close ancestry. Organisms can provide useful models for investigating the way in which genes have their effect. Provide vital information for vaccines. In short, bioinformatics allows the comparison of genomes in different organisms to investigate evolutionary relationships. At one level, sequence data confirms the division of life into three domains. At another level it shows the similarities between genes of organisms with very different phenotypes and the ways of life.

Answer 34

This term is often used to refer to the process of genetic modification by means that are not possible using selective breeding. It involves the removal of a gene or genes from one organism and placing them into another. At one extreme this involves transferring genes between species. But this can also involve taking a gene from an individual of a species and transferring it into other individuals of the same species.

Answer 35

The DNA corresponding to a gene is obtained in one of a number of ways and inserted into a vector, such as a virus, plasmid, bacterial artificial chromosome (BAC) or liposome, which is used to transfer the gene into host cells. Alternatively, the DNA can be inserted directly into cells without using a vector. The genetic code is universal so it is possible to make transfers between widely different species; for example, from a human into a bacterium. All cells give this genetic code the same meaning so the protein originally encoded by the gene will be the same protein that is produced in any cell to which it is transferred.

Answer 36

Modifying bacteria and eukaryotic cells to make special chemicals that are only produced in small quantities by other methods Making crop plants resistant to diseases, pests and herbicides Making livestock resistant to diseases and pests Improving the yields from crop plants and livestock Improving the nutritional qualities of crop plants Modifying animals to make human proteins for medicines that are difficult to obtain by other methods. Modifying bacteria so they absorb and metabolise toxic pollutants.

Answer 37

An enzyme that catalyses the reverse of transcription. The enzyme uses RNA as a template to synthesise a polynucleotide using deoxynucleotide triphosphate molecules (dNTPs). The complementary DNA strand that is produced has a base sequence that is complementary t the sequence of bases on RNA. When a molecule of mRNA isolated from the cytoplasm of cells is used as the template,the cDNA has the same base sequence as the coding strand of the nuclear DNA from which it was formed in transcription. The advantage of this is that the cDNA has no introns, only exons.

Answer 38

Are used to cut genes from lengths of DNA. They cut across both strands of DNA at specific sites known as restriction sites (as they are restricted to cut only at these sites).

Answer 39

From bacteria that are too dangerous or difficult to culture so the genes that code for them are removed and inserted into safer strains of bacteria that are modified to produce high quantities of them.

Answer 40

Each restriction site has a specific nucleotide sequence which is palindromic, so it reads the same in the 5’ to 3’ direction as in the 3’ to 5’ direction. They are present in bacteria to defend against attack by viruses; this is called restricting an infection by viruses.

Answer 41

They cut DNA to give it free, unpaired ‘ends’. They will form base pairs with complementary sequences of bases. This is how a gene cut by restriction enzyme be inserted into a plasmid or into a viral DNA. Others cut straight across DNA to give blunt ends.

Answer 42

Often necessary to produce large quantities of it. This is done by inserting the gene into bacteria and using the bacteria to replicate the gene. Done by inserting the gene into a vector, such as plasmid or a virus, which then takes it into the bacterium. The plasmid or viral DNA must have the same restriction site as the gene so that the same restriction enzyme can cut it to give a short length of complementary bases. DNA cut by restriction enzyme can have a length of nucleotides added to give it sticky ends. Copies of the gene are mixed with plasmid. Some plasmids will take up the gene by complimentary base pairing of sticky ends; other plasmids will simply reform without taking up the gene. Hydrogen bonding between sticky ends attaches the two and the enzyme ligase is added to form the covalent phosphodiester bonds of the sugar-phosphate backbone of DNA.

Answer 43

These are produced once the gene is inserted into a vector. The host bacteria are treated with calcium ions, then cooled and given an electric shock to increase the chances of plasmids passing through the cell surface membrane. This process is called electroporation. The bacteria are now described as transformed because they contain foreign DNA.

Answer 44

Some plasmids take up the foreign gene and some bacteria do not take up plasmids. There are variety of ways to identify the transformed bacteria from those that do not have the recombinant plasmids. Similar techniques are used to identify eukaryotic organisms that have been genetically modified.

Answer 45

Antibiotic-resistance genes: the foreign genes are inserted into these resistance genes so that they cannot be expressed; transformed cells are therefore sensitive to the antibiotic and untransformed cells are not the gene for green fluorescent protein (CFP), which is a small protein that emits bright green fluorescence in blue or UV light The gene for beta glucuronidase (GUS), an enzyme from E.coli that converts colourless substrates into coloured products; it is used in plants to indicate that they have been genetically engineered.

Answer 46

It copies the plasmids; the bacteria then divide by binary fission so that each daughter cell has several copies of the plasmid. The bacteria transcribe and may translate the foreign gene. If the bacteria produce a foreign protein they are described as transgenic. For transcription to occur in the host organism a promotor must be inserted with the foreign gene.

Answer 47

The length of DNA that is constructed from promoter, foreign gene and marker gene, and maybe other genes as well, is known as a gene construct or just construct

Answer 48

Bacteria can make many copies of genes very quickly. This gives many copies that can be used in research. Alternatively, the bacteria are grown in large quantities to make specific proteins, such as enzymes for the food industry.

Answer 49

It is not possible for the bacteria to carry out the complex post-translation processes of eukaryotic cells to cut, fold and modify proteins by adding sugars. As a result, genetic engineers might use yeast, plant or animal cells as the host cells for the production of proteins.

Answer 50

Have simple nutritional requirements Large volume of product are produced The production facilities do not require much space Processes can be carried out almost anywhere in the world

Answer 51

The first GM crop plants had genes to improve cultivation by incorporating pest and herbicide resistance. Pest resistance reduce losses to insect pests. Herbicide resistance allows farmers to spray herbicides during the growth of the crop to kill weeds that compete with the crop. A recent development involves crops that are designed to improve human health. Ex: GM rice known as Golden rice

Answer 52

Soy bean plants are highly susceptible to grazing by a variety of insect pests. Losses to pests are estimated to run into billions of dollars each year. In response, a biotechnology company inserted a gene for pest resistance into its herbicide-variety known as Roundup Ready. The plants are modified to express a gene from the bacterium Bacillus thuringiensis which codes for a toxin (Bt toxin) that is poisonous to insect pests. The toxin binds to receptors located on the microvilli of epithelial cells in the gut of the larvae. It inserts into the membrane, causing formation of pores or ion channels and a water potential imbalance that eventually kills the insects.

Answer 53

Many biotechnology companies have patented the genetic modifications that they have developed. To gain an economic return for the years of investment in research and development, they charge farmers a higher price for GM seed than for non-GM seed. Farmers who grow GM crops such as Roundup Ready must buy the herbicide Roundup from the same company. Increasingly new GM varieties will become available that combine that feature with others, such as pest resistance, high yield and efficient use of water.

Answer 54

Antibiotic resistance genes used to identify GM organisms could ‘escape’ and be transferred to pathogenic organisms, making it impossible to treat infections using such antibiotics Herbicide-resistance genes could be transferred in pollen to weed species and lead to development of ‘superweeds’ that are resistant to herbicides. An increase in infection by plant pathogens has been correlated with an increase in the use of glyphosate herbicides on herbicide-resistant crops; this has been particularly the case with alfalfa in the USA. Foreign genes could be transferred to wild relatives of crop plants, or to non-GM and organic crops, so changing their genomes; this may ‘pollute’ those species. Foreign genes could mutate once they have escaped and have unforeseen consequences; their insertion into the genome could activate or silence genes Making crops resistant to herbicides enables farmers to use more herbicides than on non-GM crops; this increases costs to farmers Farmers cannot keep seed for sowing for the following crop as GM crops do not ‘breed true’; this favours large-scale commercial farmers and not many farmers in developing countries Genetic material from viruses used in genetic engineering could become incorporated with natural viruses so that an animal virus can infect humans or other animals or become more harmful; entirely new pathogenic viruses may evolve by this means We are dependent on seven types of grain-producing crop plant, each of which is becoming genetically uniform and losing its genetic diversity; this means that the major food sources of the planet are increasingly dependent on our manipulation of their genomes to meet future environmental challenges.

Answer 55

The growth of GM crops needs to be approved by authorities in the EU. About half the countries, including France and Germany, have banned the growth of GM crops. There is only one GM crop that has been approved for growth in the EU that is commercially grown. This is a type of maize that is resistant to the European corn borer, Ostrinia nubilalis.

Answer 56

GM animals are divided into two groups. The first group has been genetically modified to enhance overall performance and the whole animal will become available for the food market. No GM animals have been approved by regulatory bodies for human food. The second group of GM animals are transformed to produce specific substances in milk, egg or blood- so these can be used to treat diseases or serve as a medical research models.

Answer 57

Sheep and goats have been genetically modified to produce human proteins in their milk Human antithrombin is produced by goats: this protein is used to stop blood clotting Human alpha-antitrypsin is produced by sheep: this is used to treat people with emphysema.

Answer 58

The use of livestock to produce pharmaceuticals | The animals involved are known as ‘biopharm’ animals.

Answer 59

Insulin production for the treatment of diabetes Human growth hormone to encourage growth in children with a deficiency of this hormone Thyroid-stimulating hormone for treatment of thyroid cancer Factors 8 and 9 for treating people who have a deficiency of one or other of these blood-clotting proteins Vaccines Monoclonal antibodies

Answer 60

Restriction enzymes and ligases

Answer 61

Large scale production Cheaper prices of the substances concerned Production does not rely on other factors such as the availability of insulin from dead animals.

Answer 62

This has been modified to investigate its metabolism, drug resistance and the ways in which it causes disease. It has been modified so that it will grow more rapidly, making it easier to study in vitro. Such research also gives clues to ways in which vaccines can be developed and drugs produced for treatment.

Answer 63

Have been genetically modified to deliver genes in gene therapy. Adenovirus is a very suitable vector as it will infect human and other mammalian cells; it is not species- or cell-type-specific. These viruses have had two genes removed so that they do not replicate once they infect host cells. This removal gives space in the viral genome to insert a gene or genes.

Answer 64

The recombinant human adenovirus type 5 expresses enhanced green fluorescent protein (GFP) under the control of a promoter. GFP is a protein originally isolated from Aequorea victoria, a bioluminescent jellyfish that fluoresces green when exposed to blue or UV light. Enhanced GFP is a GFP mutant with improved fluorescence and stability which is used as a marker. Therefore, a green glow in UV light indicates that the virus has successfully delivered its genes.

Answer 65

To deliver the gene for a decapeptide hormone known as TMOF into the cells of crop plants. The GM viruses are sprayed on crop plants and invade cells. The host cells transcribe and translate the gene to produce TMOF that inhibits the production of the enzyme trypsin by insect pests without having serious effects on the host plant. The DNA sequence for TMOF was combined with that for TMV coat protein so that the decapeptide and coat protein are translated as one molecule. Trypsin cuts the peptide bond that joins to the two together so releasing active TMOF which then inhibits further production of trypsin by insects. After harvest,bthe leaves of the GM plants can be processed into a powder to be used as a spray to protect against insects such as mosquitoes.

Answer 66

Proteins do not have to be extracted from animal sources or by collecting blood from many donors. The disadvantage of using bacteria to produce human proteins is that bacteria do not modify their proteins in the same way. It is much better, therefore, to use eukaryotic cells to produce human proteins.

Answer 67

Transgenic microorganisms do not compete well in the natural environment as they are engineered to produce substances that give them no advantage and they require much energy that is not readily available to produce these substances. Containment facilities, such as filters on air conditioning and air locks on doors are fitted to prevent the escape of organisms Lethal genes are added to the microorganisms so that they die if removed from the conditions of the culture.

Answer 68

This occurs when DNA is cut in specific places, where the genome can be edited. One application of this system is to alter specific genes without changing other parts of an animal’s genome. So, researchers can improve crops and livestock by replicating small genetic variations found naturally in different varieties of the same species.

Answer 69

This is an application of the principle of genetic engineering. It involves several different methods to correct a genetic ‘error’. Replacing a mutated gene that causes disease with a functioning version of the gene Introducing a new gene into the body to help fight a disease Repairing mutated genes by using an enzyme to edit the DNA and inserting a functioning gene. Inactivating or ‘knocking out’, a mutated gene that is not functioning properly.

Answer 70

Somatic cell gene therapy: somatic cells are body cells; all the cells in the body except gamete-forming cells and gametes. These cells all die when the individual dies so any genetic changes are not passed on to the next generation. However, gene therapy can be a treatment and maybe even a cure, for genetic conditions. Long-term treatments are possible by inserting a functioning allele into stem cells, such as those in bone marrow. It is most successful in cells that have a long life, such as those in the retina. It is less successful in cells that are replaced every few days, such as those in the airways and in the epithelium lining the gut. Germ-line gene therapy: the germ line is the term given to cells that differentiate to form gametes. In mammals, the germ line forms fairly early in development and this group of cells populates the gonads: the ovaries and testes. These cells increase in number by mitosis and then some will divide by meiosis to form gametes. If a correctly functioning allele will divide into a fertilised egg, it means that all the cells formed by mitosis from that cell, once fertilised, will be genetically altered. The change is permanent and will be inherited by future generations.