Manipulating genomes

Question 1

Define DNA sequencing

Answer 1

Working out the sequence of bases in a strand of DNA

Question 2

Define terminator bases

Answer 2

Modified versions of the four nucleotide bases which stop DNA synthesis when they are included

Question 3

Define high-throughput sequencing

Answer 3

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

Question 4

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

Answer 4

1. DNA is mixed with a primer (DNA polymerase), an excess of normal nucleotides and terminator bases
2. The mixture is placed in a thermal cycle that rapidly changes temperature at programmed intervals in repeated cycle
   - 96 degrees- double-stranded DNA is broken into single strands
   - 50 degrees- primers anneal to the DNA strand
   - 60 degrees- DNA polymerase starts to build up new DNA strands by adding nucleotides with complementary base to the single strand DNA template
3. Each time a terminator is incorporated instead of a normal nucleotide the synthesis of DNA is terminated as no more bases can be added. Terminators are in lower amounts and added at random so you get many DNA fragments of different lengths depending on where they were added during the process
4. After many cycles all the possible DNA chains will be produced and the DNA fragments are separated according to their length by capillary sequencing- like gel electrophoresis in minute capillary tubes.
5. The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Lasers detect the different colours and thus the order of the sequence.
6. The order of bases in the capillary tube shows the sequence of the new, complementary strand of DNA which has been made. This is used to build up sequence of the original DNA strand
7. The data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding areas of overlap between them.

Question 5

What is the purpose of DNA sequencing

Answer 5

1. Identify genes or parts of the genome that code for specific characteristics
2. Medical researchers want to identify regions that are linked with particular diseases

Question 6

Describe the reasons for developing new DNA sequencing technologies.

Answer 6

1. More efficient- a lot quicker to use high-throughput sequencing
2. Cheaper- so more can be sequenced

Question 7

Define bioinformatics

Answer 7

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

Question 8

Define computational biology

Answer 8

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

Question 9

Define genome

Answer 9

All of the genetic material of an organism

Question 10

Define DNA barcoding

Answer 10

Technique that identifies particular sections of the genome that are common to all species but vary between them so comparisons can be made. The aim is to identify different species genome.

Question 11

Define proteomics

Answer 11

It is the study of amino acid sequencing of an organism’s entire protein complement

Question 12

Define synthetic biology

Answer 12

The design and construction of novel biological pathways, organisms or devices or the redesign of existing natural biological systems

Question 13

Define epidemiology

Answer 13

Epidemiology is the study and analysis of the distribution and determinants of health and disease conditions in defined populations.

Question 14

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

Answer 14

1. Genome analysis provides scientists with another tool to aid in species identification, by comparison to a standard sequence for the species.
2. DNA barcoding

Question 15

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

Answer 15

1. Computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vulnerable
2. But scientists increasingly recognise that apart from a few relatively rare genetic diseases caused by changes in a single genes, that our genes work together to affect our physical characteristics, out physiology, and our likelihood of developing certain diseases.

Question 16

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

Answer 16

It enables:

1. Doctors to find out the source of an infection e.g bird flu or MRSA in hospital
2. Doctors to identify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helpful prevent the spread of antibiotic resistance.
3. Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics
4. Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccine.

Question 17

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

Answer 17

1. DNA sequences of different organisms can be compared
2. Because the basic mutation rate of DNA can be calculated, scientists can calculate how long ago two species diverged from a common ancestor
3. It allows scientists to build up evolutionary trees with an accuracy they have had before.

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

1. Traditionally it was thought that genes code for a particular protein but we know there are a very different number of coding genes and number of unique proteins.
2. The DNA sequence of the genome should enable you to predict the sequence of the amino acids in all of the proteins it produces
3. The evidence is that the sequence of the amino acids is not always what would be predict 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 or relatively major genetic modification of an entire organism
2. Use of biological systems or parts of biological systems in industrial contexts e.g. use of fixed or immobilised enzymes and the production of drugs from microorganisms
3. The synthesis of new genes to replace faulty genes e.g developing treatments for cystic fibrosis
4. The synthesis of an entire new organisms e.g scientists created an artificial genome which successfully replaced the original genome in a bacterium

Question 20

Define DNA profiling

Answer 20

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

Question 21

Define exon

Answer 21

Regions of coding DNA or RNA

Question 22

Define intron

Answer 22

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

Question 23

Define locus

Answer 23

The location of a gene (or of a significant sequence) on a chromosome

Question 24

Define variable number tandem repeat

Answer 24

(VNTRs) Short sequences of DNA that are repeated many times and they vary greatly in number among individuals

Question 25

Define minisatellite

Answer 25

A region where a sequence of 20-50 base pairs will be repeated from 50 to several hundred times. These occur at more than 1000 locations in the human genome and are also known as VNTRs

Question 26

Define microsatellite

Answer 26

A smaller region of just 2-4 bases repeated only 5-15 times. Also known as short tandem repeats (STRs)

Question 27

Define short tandem repeats

Answer 27

A smaller region of just 2-4 bases repeated only 5-15 times.

Question 28

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

Answer 28

1. 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
2. Only identical twins will have an identical satellite pattern

Question 29

Name the 5 main stages in DNA profiling

Answer 29

1. Extracting the DNA
2. Digesting the sample- restriction endo-nucleases cut the DNA into fragments
3. Separating the DNA fragments- Separated using gel electrophoresis and then Southern blotting
4. Hybridisation- DNA probes are added to label the fragments. Radioactive probes attach to specific fragments
5. Seeing the evidence

Question 30

Describe the extracting DNA step in DNA profilling

Answer 30

1. DNA must be extracted from a tissue sample
2. Large samples used to be needed
3. But now using a technique called polymerase chain reaction, the tiniest fragment of tissue can give scientists enough DNA to develop a profile

Question 31

Describe the digesting the sample step in DNA profilling

Answer 31

1. The strands of DNA are cut into small fragments using special enzymes called restriction endonucleases
2. Different endonucleases cut DNA at a specific nucleotide sequence, known as a restriction site or recognition site
3. All restriction endonucleases make two cuts, one through each strand of DNA double helix
4. Restriction endonucleases give scientists the ability to cut the DNA strands at defined points in the introns
5. They use a mixture of restriction enzymes that leave the repeating units or satellites intact, so the fragments at the end of the process include a mixture of intact mini and microsatellite regions.

Question 32

Describe the separating the DNA fragments step in DNA profilling

Answer 32

1. Gel electrophoresis- a technique that utilises the way charged particles move through a gel medium under the influence of an electric current.
2. The gel is then immersed in alkali in order to separate the DNA double strands into single strands
3. The single-stranded DNA fragments are then transferred onto a membrane by Southern blotting

Question 33

Describe the hybridisation step in DNA profilling

Answer 33

1. Radioactive or fluorescent DNA probes are now added in excess to the DNA fragments on the membrane.
2. DNA probes are short DNA or RNA sequences complementary to a known DNA sequence.
3. They bind to the complementary strands of DNA under particular conditions of pH and temperature.
4. DNA probes identify the microsatellite regions that are more varied than the larger minisatellite regions.
5. The excess probes are washed off

Question 34

Describe the seeing the evidence step in DNA profilling

Answer 34

1. If radioactive labels were added to the DNA probes, X-ray images are taken of the paper/ membrane
2. If fluorescent labels were added to the DNA probes, the paper/ membrane is placed under UV light so the fluorescent tags glow- most common method today
3. The fragments give a pattern of bars which is unique to every individual except identical twins

Question 35

Describe how to separate nucleic acid fragments by electrophoresis

Answer 35

1. DNA fragments are put into wells of agarose gel strips which also contain a buffering solution
2. DNA fragments of known length are used to provide a reference for fragment sizing
3. When an electric current is passed through the electrophoresis plate, the DNA fragments in the wells at the cathode end move through the gel towards the positive anode
4. This is due to the negatively charged phosphate groups in the DNA fragments
5. Rate of movement depends on the mass or length of the fragments- gel has mesh structure- smaller fragments can move through more easily than larger fragments. So smaller fragments move further.
6. It is put in alkaline buffer solution to denature the DNA fragments. The two DNA strands of each fragment separate, exposing the bases

Question 36

What is the technique called Southern blotting

Answer 36

1. The DNA strands produced from gel electrophoresis are transferred to nitrocellulose paper or a nylon membrane, which is placed over the gel.
2. The membrane is covered with several sheets of dry absorbant paper, drawing the alkaline solution containing the DNA through the membrane by capillary action.
3. They are transferred in precisely the same relative positions as they had on the gel. They are then fixed in place using UV light or heated at 80 degrees

Question 37

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

Answer 37

1. Forensic science- PCR and DNA profiling is performed on traces of DNA left at the crime scene. The DNA profile is compared to that of a sample taken from a suspect or can be identified on the criminal DNA database
2. DNA profiling is also used to prove paternity of a child- used in immigration cases
3. Identify species to which an organism belongs and identify evolutionary relationships between different species
4. Identify individuals who are at risk of developing particular diseases. Certain non-coding microsatellites, or the repeating patterns they make have been found to be associated with an increased risk of a particular disease

Question 38

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

Answer 38

1. The DNA sample to be amplified, an excess of the four nucleotide bases, small primer DNA sequences and the enzyme DNA polymerase are mixed in a vial that is placed in a PCR machine
2. Separating the strands- The temperature of the PCR machine is increased to 90-95 degrees 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 temperature is decreased to 55-60 degrees and the primers bind to the ends of the DNA strands. They are needed for the replication of the strands to occur.
4. Synthesis of DNA- The temperature is increased again to 72-75 degrees for at least 1 minute, this is the optimum temperature for DNA polymerase.
5. DNA polymerase adds bases to the primer, building up complementary strands of DNA and so produce double-stranded DNA identical to the original sequence. 6. The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria found in hot springs.

Question 39

Define DNA primer

Answer 39

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

Question 40

Define gene therapy

Answer 40

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

Question 41

Define somatic cell gene therapy

Answer 41

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

Question 42

Define germ line cell gene therapy

Answer 42

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

Question 43

Describe the principle of somatic gene therapy in medicine

Answer 43

1. Replacing the mutant allele with a healthy allele in affected somatic cells.
2. However it is only a temporary solution for the treated individual. The healthy allele will be passed on every time a cell divides by mitosis.
3. But somatic cells have a limited life and are replaced from stem cells which will have the faulty allele.
4. A treated individual will still pass the faulty allele on to any children they have

Question 44

Describe the principle of germ line gene therapy in medicine

Answer 44

1. Insert healthy allele into the germ cells usually the eggs- or into a embryo immediately after fertilisation (as part of IVF).
2. The individual would be born healthy with a normal allele in place and would pass it on to their own offspring
3. Such therapy has been successfully done with animal embryos but is illegal for human embryos in most countries
4. Concerns is that the potential impact of intervention in germ cells is unknown
5. And human rights of unborn embryo
6. And ethical concern that the technology might eventually be used to enable people to choose desirable characteristics.

Question 45

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

Answer 45

1. People have regained some vision
2. Immune disease
3. Leukaemias
4. Myelomas
5. Haemophilia