Genome Manipulation

Question 1

Define the term “DNA sequencing”.

Answer 1

Working out sequences of bases in a strand of DNA.

Question 2

Define the term “terminator bases”.

Answer 2

Modified version of four nucleotide bases which stop DNA synthesis when they are included.

Question 3

Define the term “high-throughput sequencing”.

Answer 3

New methods of DNA sequencing that are automated, very rapid and much cheaper than original techniques.

Question 4

Describe the steps in DNA sequencing (the capillary method).

Answer 4

1) DNA mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases.
2) The mixture is placed in a thermal cycler that rapidly changes temp at programmed intervals. At 96oC the double stranded DNA separates into single strands (H-bonds between bases broken) and at 50oC the primers anneal (attach) to the DNA strand.
3) At 60oC DNA polymerase starts to build up new DNA strands by adding nucleotides with complementary bases to the single-strands of DNA.
4) Each time a terminator base is added instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added. Chain terminators bind at random so this results in many different size fragments of DNA. After many cycles, all of the possible DNA chains will be produced with reaction stopped at every base.
5) The DNA fragments are then separated according to their length by capillary sequencing. The fluorescent markers on each base are used to identify the final base on each fragment. Lasers detect the different colour and thus the order of the sequence.
6) The order of bases in the capillary tube shows the base sequence of the DNA strand which is complementary to the original strand of DNA.
7) Data from the sequencing process is fed into a computer that the reassembles the genomes by comparing all the fragments and finding the overlaps. This allows us to assemble the entire genome.

Question 5

What is the purpose of genome sequencing?

Answer 5

Medical researches want to identify regions of the genome that are linked with particular diseases.
Can help us to discover evolutionary links.

Question 6

Describe the reasons for developing new DNA sequencing technologies.

Answer 6

• More efficient, very fast.
• Cheaper.
• 3 billion base pairs of the human genome can be sequenced in just days.

Question 7

Define the term “bioinformatics”.

Answer 7

The development of software and computing tools needed to analyse and organise raw biological data.

Question 8

Define the term “computational biology”.

Answer 8

The study of biology using computational techniques to analyse large amounts of data.

Question 9

Define the term “genome”.

Answer 9

All the genetic material of an organism.

Question 10

Define the term “DNA barcoding”.

Answer 10

The identification of particular sections of the genome that are common to all species but vary between them, so comparisons can be made.

Question 11

Define the term “synthetic biology”.

Answer 11

The design and construction of novel biological pathways or devices or the re-design of existing natural biological systems.

Question 12

Define the term “proteomics”

Answer 12

The study of amino acid sequencing of an entire an organism’s protein complement.

Question 13

Define the term “epidemiology”.

Answer 13

The study of how often diseases occur in different groups of people and why.

Question 14

Explain why new DNA sequencing methods are allowing genome-wide comparisons between individuals and between species.

Answer 14

Genome analysis provides scientists with another tool to aide in species identification, by comparison to a standard sequences for a species.

Question 15

Explain why the comparison of many human genomes may help the understating and treatment of human illness.

Answer 15

• Reveals patterns in the DNA and the diseases to which we are vulnerable.
• Enormous implications for the field of medicine.
• Identify changes in gene which cause a genetic disease and our likelihood of developing certain disease.

Question 16

Describe 4 reasons why scientists may want to analyse the genomes of pathogens.

Answer 16

• Find the source of infection.
• Identify anti-biotic resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping prevent the spread of antibiotic resistance.
• Scientists can track the outbreak of a potentially serious disease.
• Identify regions in the genome of pathogens that may be useful targets in the development of new drugs.

Question 17

Describe how DNA sequencing allows scientists to identify the evolutionary relationships between species.

Answer 17

• DNA sequences of different organisms can be compared.
• Basic mutation rate of DNA can be calculated so scientists can calculate how long ago two species diverged from a common ancestor.
• Build up evolutionary trees.

Question 18

Explain why, in theory, knowing a DNA sequence should allow you to identify the sequence of amino acids in the protein that the DNA sequence codes for. Also, explain why, in practice, this doesn’t always provide the correct sequence of amino acids in the protein.

Answer 18

• Because we traditionally thought that genes code for a particular protein, but we now know that the number of coding genes in the human DNA is very different the number of unique proteins.
• The expressed sequence of amino acids is not always what would be predicted from the genome sequence alone.
• Some genes can code for many different proteins.

Question 19

Describe 4 techniques that could be classified as “synthetic biology”. Describe the role of DNA sequencing in each technique.

Answer 19

1) Genetic engineering - may involve a single change in a biological pathway or relatively major genetic modification of an entire organism.
2) Use of biological systems or parts of biological systems in industrial contexts, for example, the use of fixed or immobilised enzymes and the production of drugs from microorganisms.
3) The synthesis of new genes to replace faulty genes, r.g the faulty genes that cause cystic fibrosis.
4) The synthesis of an entire new organism, i.e. creating an artificial genome.

Question 20

Define the term “DNA profiling”.

Answer 20

Producing an image of the patterns in the non-coding DNA of an individual.

Question 21

Define the term “exon”.

Answer 21

Regions of coding DNA or RNA.

Question 22

Define the term “intron”.

Answer 22

Regions of non-coding DNA or RNA that are removed from mRNA before it is translated.

Question 23

Define the term “locus”.

Answer 23

The location of a gene on a chromosome.

Question 24

Define the term “variable number tandem repeat” (VNTR).

Answer 24

• Short sequences of DNA repeated many times which vary greatly in number between individuals.

Question 25

Define the term “minisatellite”.

Answer 25

A region where a sequence of 20-50 base pairs (satellite DNA) will be repeated from 50 to several hundred times.
These occur at more than 1000 locations in the human genome.

Question 26

Define the term “short tandem repeat”.

Answer 26

• A microsatellite is a smaller region of just 2-4 base pairs repeated only 5-15 times.
• Known as short tandem repeats.

Question 27

Define the term “microsatellite”.

Answer 27

• A smaller region of DNA just 2-4 base pairs long and repeated only 5-15 times.

Question 28

Describe how STRs vary and why they can be used to identify individuals.

Answer 28

• These satellites always appear in the same positions on the chromosomes, but the number of repeats of each mini/micro satellites varies between individuals, as different lengths of repeats are inherited from both parents.
• Only identical twins will have an identical satellite pattern.
• The more closely related you are to someone, the more likely you are to have a similar satellite pattern.

Question 29

Name the 5 main stages in DNA profiling.

Answer 29

1) Extraction DNA.
- DNA is extracted from the sample.
2) Digesting the sample.
- Restriction endonucleases cut the DNA into fragments.
3) Separating the DNA fragments.
- DNA fragments are transferred from the gel to the nylon membrane in a process known as southern blotting.
4) Hybridisation.
- DNA probes are added to label the fragments.
- These radioactive probes attach to specific fragments.
5) Seeing the evidence.
- Membrane with radioactively labelled DNA fragments is place onto an x-ray film.
- Development of the x-ray film reveals dark bands where the radioactive or fluorescent DNA probes have attached.

Question 30

Described the extraction of DNA in DNA profiling (Stage 1).

Answer 30

• DNA is extracted from tissue sample. PCR used to amplify small tissue sample.

Question 31

Describe how the DNA sample is digested in DNA profiling (stage 2).

Answer 31

• Strands of DNA are cut into small fragments using special enzymes called restriction endonucleases.
• Different restriction endonucleases cut DNA at a specific nucleotide sequences, known as a restriction site.
• All restriction endonucleases make two cuts, once through each strand of the double-stranded helix.
• Restriction endonucleases have given scientists the ability to cut the DNA strands at defined point in introns. They use a mixture of restriction enzymes that leave repeating units/satillites intact so the fragments at the end include a mixture of intact mini- and micro-satellite regions.

Question 32

Describe how the DNA sample is separate in DNA profiling (stage 3).

Answer 32

• Gel electrophoresis is used to separate the DNA fragments.
• This produces a clear and recognisable pattern (a DNA profile).
• A charge is applied across the gel and then is immersed in akali in order to separated the DNA double strands into single strands.
• The single stranded DNA fragments are then transferred onto a membrane by southern blotting.

Question 33

Describe the hybridisation stage of DNA profiling (stage 4).

Answer 33

• Radioactive or fluorescent DNA probes are now added in excess to the DNA fragments on the membrane.
• DNA probes are short sections of DNA/RNA sequences complementary to a known DNA sequence.
• They bind to complementary strands of DNA under particular conditions of PH and temperature.
• This is called hybridisation.
• DNA probes identify the micro-satellite regions that are more varied than the the larger mini-satellite regions.
• The excess probes are washed off.

Question 34

Describe the ‘seeing the evidence’ stage of DNA profiling (stage 5).

Answer 34

• If radioactive labels were added to the DNA probes, x-ray images are taken of the paper/membrane.
• If fluorescent labels were used, the paper/membrane is placed under UV light so that the fluorescent tags glow (most common).
• The fragments give a pattern of bars - then DNA profile - which is unique to every individual except identical siblings.

Question 35

Describe the separation of nucleic acid fragments by electrophoresis.

Answer 35

• DNA fragments are put into wells of agarose gel strips, which also contain a buffering solution to maintain a constant PH.
• An electric current is passed through the gel plate. The DNA fragments are negatively charged so they move across the gel towards the positive electrode (the anode).
• The gel has a mesh-like structure that resists the movement of molecules. Smaller fragments can move through the mess more easily than larger fragments, so they small fragments move furthest. Fragments separated by size.
• Charge is switched off when small fragments reach the anode end of the gel.
• The gel is then placed in an alkaline buffer solution to denature the DNA fragments. The two DNA strands of each fragment separate, exposing the bases (for attachment of probes).

Question 36

What is the process of southern blotting?

Answer 36

• Single stranded fragments of DNA produced from gel electrophoresis are transferred to a nitrocellulose paper or a nylon membrane, which is placed over the gel.
• The membrane is covered with several sheets of dry absorbent paper, drawing the alkaline solution containing the DNA through the membrane by capillary action.

Question 37

Suggest how DNA profiling can be used to answer questions about identity in different situations.

Answer 37

• Used in forensic science. Traces of DNA left at a crime scene can be obtained from blood, semen, salvia, hair and skin cells. Resulting DNA profile can be compared to that of a suspect. Prove either guilt or innocence.
• Used also to prove paternity of a child.
• Can be used to demonstrate evolutionary relationships between species.
• Identifying individuals who are at risk of developing particular diseases i.e. the presence of certain non-coding microsatellites can be associated with an increased risk of particular diseases.

Question 38

Explain how to determine whether a person is homozygous or heterozygous for a particular STR (and gender) given appropriate information.

Question 39

Describe how DNA can be amplified using the polymerase chain reaction (PCR).

Answer 39

1) An excess of four nucleotide bases A, T, C and G, smaller DNA primers and the enzyme DNA tap polymerase are added to PCR machine.
2) Separating strands - temp in PCR machine raised to 90oC for 30 seconds. This denatures the DNA by breaking the hydrogen bonds holding the DNA strands together so they separate.
3) Annealing of the primers - the temp is decreased to 55-60oC and the primers bind (anneal) to the ends of the DNA strands. They are needed for the replication of strands to occur.
4) Synthesis of DNA - the temperature is increased again to 72-75oC for at least one minute. This is the optimum temp for DNA polymerase, which adds bases to the primer, building up complementary strands of DNA and so producing double-stranded DNA helix identical to the original sequence.

– DNA tap polymerase is a special strain which is very resistant to heat.

Question 40

Define the term “DNA primer”.

Answer 40

A primer is a short strand of RNA or DNA that serves as a starting point for DNA synthesis.

Question 41

Define the term “genetic engineering”.

Answer 41

The modification of the characteristics of an organism by manipulating its genetic material.

Question 42

Define the term “transgenic”.

Answer 42

An organism that contains a gene from another organism.

Question 43

Define the term “vector”.

Answer 43

A means of inserting from one organism into the cells of another.

Question 44

Define the term “recombinant DNA”.

Answer 44

DNA that contains genetic material from two sources.

Question 45

Define the term “transformation”.

Answer 45

The process where a plasmid with recombinant DNA must be transferred into the host cell.

Question 46

Define the term “electroporation”.

Answer 46

The use of a very tiny electric current to transfer genetically engineered plasmids into bacteria or to get DNA fragments directly into Eukaryotic cells.

Question 47

Define the term “marker gene”.

Answer 47

A gene used to determine if a nucleic acid sequence has been successfully inserted into an organism’s DNA.

Question 48

Define the term “DNA ligase”.

Answer 48

Facilitates the joining of DNA strands together by catalysing the formation of phosphodiester bonds.
– Only in the formation of recombinant DNA!! The rest of the time DNA polymerase does this job.

Question 49

Define the term “sticky ends”.

Answer 49

An end of DNA double helix in which a few unpaired nucleotides of one strand extend beyond the other.

Question 50

Describe the principles of genetic engineering.

Answer 50

• Isolating a desired gene from one organism and placing it into another organism, using a vector.
• The organisms may be of the same species/ different species.
• Organism that carries a gene from another organism is ‘transgenic’ or GMO (genetically modified organism).

Question 51

Describe how a desired gene can be isolated using restriction endonucleases.

Answer 51

• Most common technique is to use restriction endonucleases to cut out desired gene from DNA.
• restriction endonucleases can cut DNA at a specific base sequence so can isolate the desired gene and produce sticky ends.
• These sticky ends make it much easier for the gene to be inserted into the vector (which has been cut with the same restriction endonuclease that was used for the genetic material containing the desired gene).

Question 52

Describe how a desired gene can be isolated using reverse transcriptase.

Answer 52

• The mRNA for the desired gene is isolated.
• The enzyme reverse transcriptase can be used to produce a single strand of complementary DNA.
• This technique makes it easier to identity the desired gene because a particular cell type will make some very specific types of mRNA.

Question 53

Explain how a plasmid can be used as a vector.

Answer 53

• A plasmid is a small circular molecule of chromosomal DNA within a bacterium that can replicate independently.
• Once a plasmid gets into a new host cell it can combine with the host DNA to form recombinant

Question 54

Explain how marker genes can be used to identify organisms that have been successfully genetically engineered.

Answer 54

• Plasmids that are used as vectors are often chosen because they contain a marker gene.
• A marker gene could be antibiotic resistance, or they have been genetically modified to have fluorescence as a marker gene.
• This gene enables scientists to determine that the bacteria have taken up the plasmids, i,e by growing bacteria in media containing antibiotics (replica plating).

Question 55

Describe 4 ways in which a recombinant plasmid can be inserted into target cells.

Answer 55

The transfer of a recombinant plasmid into a host cell is called transformation. The methods:

1) Culture the bacterial cells in a calcium rich solution and increase the temp. Causes the bacterial membrane to become more permeable and plasmids can enter.
2) Electroporation, a small electrical current is applied to the bacteria. Makes membranes very porous and so the plasmids move into.
3) Electrofusion, tiny electric currents are applied to the membranes of two different cells. This fuses the cell and nuclear membranes of the two different cells to form a hybrid, containing DNA from both cells.

Question 56

What is the second marker gene and how is it useful?

Answer 56

• Plasmid vectors are usually given a second marker gene, which is used to show that the plasmid has taken up the isolated gene.
• This marker gene is often genetically engineered into the plasmid.
• The marker gene within the plasmid is cut with restriction enzyme so that the desired gene can be inserted.
• If the desired gene is inserted correctly, the marker gene will not function.
• These marker genes can be for antibiotic resistance but also fluorescence. If a bacterium does not fluoresce then it has been engineered successfully.

Question 57

What is the second marker gene and how is it useful?

Answer 57

• Plasmid vectors are usually given a second marker gene, which is used to show that the plasmid has taken up the isolated gene.
• This marker gene is often genetically engineered into the plasmid.
• The marker gene within the plasmid is cut with restriction enzyme so that the desired gene can be inserted.
• If the desired gene is inserted correctly, the marker gene will not function.
• These marker genes can be for antibiotic resistance but also fluorescence. If a bacterium does not fluoresce then it has been engineered successfully.

Question 58

What is a use of electrofusion?

Answer 58

• Important in the production of monoclonal antibodies.
• A monoclonal antibody is a combination of cell that only produces one type of antibody and a tumour cell. This means it divides rapidly in culture.
• Monoclonal antibodies are used to identify pathogens in both animals and plants, and in the treatment in a number of diseases such as some cancers.

Question 59

Describe how GM plant cells can be micro-propagated.

Answer 59

1) Cut the leaf.
2) Expose the cut leaf to bacteria carrying a weedkiller-resistance gene. Allow bacteria to deliver genes into the leaf cells.
3) Expose leaf to an antibiotic to kill cells that lack the new genes. Wait for surviving (gene-altered) cells to form a callus (mass of cells).
4) Allow callus to sprout shoots and roots.
5) The plants are transferred to soil where they can develop into fully differentiated adult plants that are resistant to weed killer.

Question 60

Describe the process of using genetic engineering to produce human insulin using bacteria.

Answer 60

1) Use restriction enzyme to cut out the insulin-producing gene.
2) Use the same restriction enzyme to cut plasmid. This produces sticky ends with are complementary to the stikcy ends of the isolated gene.
3) Sticky ends line up and anneal/ complementary base pair. DNA ligase catalyses the formation of phosphodiester bonds between the sugar and phosphate groups of the nucleotides. This forms a recombinant plasmid.
4) Mix the recombinant plasmid with bacterial host cells by the process of transformation.

Question 61

Define the term “gene therapy”.

Answer 61

The introduction of normal genes into cells in the place of missing or defective ones in order to correct genetic disorders.

Question 62

Define the term “somatic cell gene therapy”.

Answer 62

Replacing a faulty gene with a healthy allele in affected somatic cells.

Question 63

Define the term “germ line cell gene therapy”.

Answer 63

Inserting a healthy allele into the germ or into a very early embryo.

Question 64

Describe the principle of gene therapy in medicine.

Answer 64

• Replace faulty alleles with a healthy ones in order to combat diseases such as CF, haemophilia and immunodeficiency.
• They can remove the desired alleles from healthy cells or synthesize healthy alleles in the lab.

Question 65

Describe the process of somatic cell gene therapy.

Answer 65

• Replacing mutant allele with healthy allele in affected somatic cells.
• Difficulty getting healthy alleles into the affected cells, getting the engineered plasmids into the nucleus of the the cells, and difficulties in starting and maintaining expression of the healthy allele.
• Viral vectors are often used.
• The healthy allele will be passed on every time the cell divides by mitosis by eventually the treated somatic cells will die, and cells with the faulty allele will take their place. So only a temporary solution.
• A treated individual will still pass faulty allele on to children.

Question 66

Describe the process of germ line gene therapy.

Answer 66

• Insert a healthy allele into germ cells, i.e egg cells, or into an embryo immediately after fertilisation.
• The individual would be born healthy with the normal alleles, and would pass on the healthy alleles to their own offspring.

Question 67

Name 5 diseases for which successful somatic cell gene therapy treatments have been reported.

Answer 67

• leukaemia
• immune diseases
• myelomas
• retinal disease
• haemophilia