6.3 - E - Manipulating Genomes

Question 1

What does PCR stand for?
What is it and how does it work?
What is it used for?

Answer 1

The Polymerase Chain Reaction.
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies.
It’s used to make enough DNA to test multiple times (crimes, genetic profiling).

Question 2

What are DNA primers?

Answer 2

10‐20 bases of single stranded DNA used for sequencing and PCR to bind to sections of DNA so that DNA polymerase can bind. DNA polymerase can’t bind to single strands, by adding primers creates a small section of a double stranded DNA which DNA polymerase can bind to.

Question 3

What are the key steps and temperatures in PCR?

Answer 3

Denaturation ‐ 95°C
Annealing ‐ 68°C
Elongation ‐ 72°C

Question 4

What facts does PCR rely on?

Answer 4

DNA is made of 2 antiparallel backbone strands.
Each strand of DNA has a 5’ end and a 3’ end.
DNA grows only from the 3’ end.
Base pairs pair up according to complementary base pairing rules, A with T and G with C.

Question 5

How does PCR differ from DNA replication?

Give one similarity.

Answer 5

Only short sequences, of up to 10,000 base pairs, of DNA can be replicated, not entire chromosomes.
It requires the addition of primer molecules to make the process start.
A cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated.
It’s artificial DNA replication, not natural.
Both copy DNA and both require polymerase.

Question 6

Explain the steps in PCR

Answer 6

The sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and the enzyme taq DNA polymerase.
The mixture is heated to around 94 to 96°C to break the hydrogen bonds between complimentary nucleotide base pairs and thus denature the double-stranded DNA into two single strands of DNA.
The mixture is called to around 68°C, so that the primers can anneal to one end of each single strand of DNA. This gives a small section of double-stranded DNA at the end of each single+stranded molecule.
The taq DNA polymerase enzyme molecules can now binds to the end where there is double-stranded DNA. Taq polymerase is obtained from a bacterium that lives at high temperatures; 72°C is the optimum temperature.
The temperature is raised to 72°C, which keeps the DNA as single strands.
The Taq DNA polymerase catalyses the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with the primer and proceeding in the five‘ to 3‘ direction.
When the Taq DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated.
The whole process begins again and is repeated for many cycles.

Question 7

State and explain the applications of PCR

Answer 7

Tissue typing - donor and recipient tissues can be ‘typed’ to reduce risk of rejection in transplants.
Detection of oncogenes (cancer genes) - trying to find the specific mutations that caused a cancer can allow more specific medication to be given.
Forensic science - Small quantities of DNA found at a crime scene can be amplified so there is enough for DNA profiling.
Detecting mutations - DNA analysed to look for mutations that cause genetic disease (could be done in parents/embryos).
Identifying viral infections - can detect small amounts of viral DNA amongst host DNA – can be used to test for e.g. HIV.
Research - Can amplify sources of DNA from fossils etc for sequencing to study evolutionary relationships. In living species genes which are switched on or off can be studied.
Monitoring the spread of infectious diseases.

Question 8

Define electrophoresis.

What is it used for?

Answer 8

A method of separating and ordering DNA fragments or proteins based on size.
Used so that the fragments can be identified and analysed.
Used in sequencing and DNA profiling.

Question 9

Explain how the electrophoresis of DNA works

Answer 9

Small amounts of DNA can be amplified using PCR.
DNA is cut into smaller fragments using restriction enzymes. (The same restriction enzyme must be used to cut the fragments from any of the individuals involved in the identification for forensics).
The fragments are placed into the wells at the end of the gel plate where the negative electrode (cathode) will be.
The plate is immersed into a tank filled with buffer solution and an electric current is passed through the tank (1‐2 hours).
DNA is negatively charged (due to the phosphoryl groups of the sugar‐phosphate backbone) and so are attracted to the other end of the plate, where the positive electrode (anode) is, so the molecules diffuse along the gel to the other end.
The shorter fragments move further in the same period of time than the longer ones.
The banding pattern is invisible so the DNA must be stained with ethidium bromide and then viewed under UV light to observe the final banding pattern.

Question 10

Explain how the electrophoresis of proteins work

Answer 10

This is done in the same way as DNA.
Sodium dodecyl sulfate (SDS) is added to proteins to give them equal negative charge.
This means that they can be separated by molecular mass (rather than charge). This can be used to analyse proteins by mass in blood to diagnose medical conditions:
Sickle cell anaemia.
Diseases in which patients have higher levels of fetal haemoglobin than they should.

Question 11

What is a DNA probe?

What can they be labelled using?

Answer 11

A short (50-80 nucleotides) single-stranded length of DNA that is complementary to a section of the DNA being investigated.
They can be labelled using:
A radioactive marker usually with 32p in one of the phosphate groups in the probe strand. Once the probe has annealed, by complimentary base pairing, to the piece of DNA, it can be revealed by exposure to photographic film.
A fluorescent marker that emits a colour on exposure to UV light. Fluorescent markers may also be used in automated DNA sequencing.

Question 12

Probes are useful in locating specific DNA sequences, for example…

Answer 12

To locate a specific gene needed for use in genetic engineering.
To identify the same gene in a variety of different genomes is from different species when conducting genome comparison studies.
To identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition.

Question 13

What is a DNA microarray?
What does this show?
How do they work?

Answer 13

They are the fixed surface that scientists can place a number of different DNA probes on to. This can reveal the presence of mutated alleles that match the fixed probes, because the sample DNA will anneal to any complementary fixed probes.
The sample DNA must first be broken into smaller fragments and it may also be amplified using the PCR. A DNA microarray can be made with fixed probes, specific for certain sequences found in mutated alleles that cause genetic diseases, in the well.
Reference and test DNA samples are labelled with fluorescent markers. Where a test subject and a reference marker are both bind to a particular probe, the scan reveals fluorescence of both colours, indicating the presence of the particular sequence in the test DNA.

Question 14

What is DNA profiling?
What is this often used for?
How much of human DNA would be suitable for DNA profiling and why?

Answer 14

DNA profiling (also called DNA fingerprinting) is a way of identifying individuals by characteristics of their DNA.
Often this is used to compare the DNA of more than one individual.
Almost all human DNA is the same or very similar (particularly the genes
which code for proteins) so a lot of it would not be suitable for
comparisons.

Question 15

What does STR stand for?

What are they?

Answer 15

Short Tandem Repeats are loci on the genome composed 2‐10 base pairs which repeat between 5‐50 times in a row.
The number of repeats at each loci varies from person to person so we
can use these to compare the DNA of different individuals.

Question 16

Explain the procedure of DNA profiling

Answer 16

DNA is obtained from the individual - saliva, blood, hair, ancient bone.
The DNA is then digested with restriction enzymes. These enzymes cut the DNA at specific recognition sites. They will cut it into fragments, which will vary in size from person to person.
These fragments are separated by gel electrophoresis and stained. Larger fragments travelled the shortest distance in the gel.
A banding pattern can be seen.
The DNA to which the individuals is being compared is treated with the same restriction enzymes and also subjected to electrophoresis.
The banding patterns of the DNA samples can then be compared.

Question 17

How many STR loci are needed for electrophoresis?

Answer 17

About 10% of people will share the same number of repeats at any loci, so to produce a DNA profile, 13 STR loci are analysed.
The chance of two people sharing the same number repeats in each STR
at 13 separate loci is about 1 x 10^13.

Question 18

How do we find out the number of STR’s a person has at each location?

Answer 18

Electrophoresis: more repeats in STRs = larger DNA fragment = moves less far in electrophoresis.

Question 19

Define polymorphic

Answer 19

Occurring in several different forms, in particular with reference to species or genetic variation.

Question 20

Explain the steps in creating polymorphism

Answer 20

DNA obtained from all people to be compared e.g. from saliva/hair.
DNA amplified using PCR.
DNA from all people cut into different size fragments using the same restriction enzymes - DNA from different people will be different sizes because the number of repeats in the STR will vary.
DNA fragments separated based on size using electrophoresis - people to be compared are loaded into different wells.
Banding pattern examined (small fragments move further) and compared.

Question 21

State the applications of DNA profiling

Answer 21

Forensic science
Maternity and paternity testing
Studying evolutionary relationships
Analysis of disease

Question 22

List the ways in which forensic science is an application of DNA profiling. Include specific examples.

Answer 22

Convicting criminals of crimes based on DNA left at crime scenes.
Identifying body parts in fires/plane crashes.
Established innocents of many previously wrong convicted.
Identifying nazi war criminals hiding in South America.
Identifying remains found in Leicester as those of Richard III.
Identifying victims’ body parts after air crashes, terrorist attaches or other disasters.
Match profiles from descendants of those lost during WWI with the unidentified remains of the soldiers who fell on battlefields in Northern France.

Question 23

Explain how maternity and paternity tests are an application of DNA profiling

Answer 23

Half of a child’s DNA, and therefore half the STRs on a DNA profile, is from the mother and half from father.
Comparing the DNA profiles of mother, father and child can therefore establish maternity and/or paternity.

Question 24

Explain how analysis of disease is an application of DNA profiling

Answer 24

This is also known as genetic screening. Protein electrophoresis an detect the type of haemoglobin present and aid diagnosis of sickle cell anaemia. A varying number of repeat sequences for a condition such as Huntington disease are caused by STRs which repeat too many times. This can be detected by electrophoresis.

Question 25

Explain how studying evolutionary relationships is an application of DNA profiling

Answer 25

The more similar the banding pattern, the more closely related.
Eg: finding common ancestors between different species.

Question 26

Define genetic screening. Evaluate it.

Answer 26

Testing of a population to identify individuals who are at risk for a genetic disease or for transmitting a gene for a genetic disease.
+ can identify presence of disorder
+ removes uncertainty
+ allows early treatment which may improve life expectancy/Q.O.L.
+ allows informed choice about having children
+ allows IVF and embryo screening
+ allows fetal testing and termination - choice, re donation/adoption
-false, positives negatives
-only small number tests available/not available for all conditions
-presence may not result in condition
-confirmed presence gives stress/fear
-problem re, telling/testing, rest of family
-discrimination by, employers/insurers
-ethics of termination
-could increase intolerance/discrimination of disabled

Question 27

What are the ‘bands’ in a DNA profile/DNA fingerprint image?

Answer 27

DNA fragments of different lengths. They are different lengths
because they are STRs of varying numbers of repeats.

Question 28

Define DNA sequencing

Answer 28

The process working out the order of bases on a DNA molecule/gene. It’s a technique that allows genes to be isolated and read.

Question 29

Explain the development of DNA sequencing

Answer 29

Early methods (1970s) worked from mRNA, were very slow and only suitable for very short genes.
In 1975, Fred Sanger developed a method of sequencing entire genomes.

Question 30

Explain the steps in Fred Sanger’s original 1975 DNA sequencing approach

Answer 30

Single stranded DNA, DNA polymerase, primers and DNA nucleotides are mixed with one type of radioactively labelled nucleotides (once added to a sequence terminate DNA synthesis).
DNA sequences of every possible length (each terminating with a
radioactively labeled nucleotide) were generated.
These were run via gel electrophoresis to work out the sequence on the gene. The nucleotide base at the end of each fragment was read according to its radioactive label.
If the first one-base was T then the first base in the sequences is T.
If the 2-base fragments have C at the end, then the sequence is TC.
If the 3-base fragment ends with G, then the base sequence is TCG.
This was very slow and labour intensive.

Question 31

Explain the steps in cloning DNA

Answer 31

The gene to be sequenced is isolated, using restriction enzymes, from a bacterium. The DNA is then inserted into a bacterial plasmid (the vector) and then into an E. coli bacterium that, when cultured, divides many times, enabling the plasmid with the DNA insert to be copied many times. Each new bacterial contains a copy of the cancer gene. These lengths of DNA are isolated using plasmid preparation and are then sequenced.

Question 32

Explain the steps in Sanger’s updated 1986 method

Answer 32

Fluorescently labelled nucleotides replaced the radioactive ones.
All 4 types of labeled nucleotides could be placed together (rather than
separately) in a sequencing machine.
Instead of gel electrophoresis, the machine ran the different lengths of DNA through a gel in a capillary tube.
A laser scanned each length read the fluorescent base sequence as a sequence of colours (specific to each base) to reveal the sequence.
This was much faster and less labour intensive.

Question 33

How do you read a DNA strand sequence after gel electrophoresis?

Answer 33

Positive to negative

Question 34

What happened in the 2000’s?
Why are these better than previous techniques?
Give an example.

Answer 34

A range of ‘high throughput sequencing’ methods were developed.
These are much faster at sequencing whole genomes than the Sanger method and cheaper (although more errors tend to be made).
Pyrosequencing.

Question 35

When was pyrosequencing developed?
How is it different from Sanger’s method?
What does it involve?
Explain the method.

Answer 35

1996. It uses sequencing by synthesis, not by chain termination (as used by Sanger).
      It involves synthesising a single strand of DNA, complementary to the strand to be sequences, one base at a time, whilst detecting, by light emission, which base was added at each step.

Question 36

Explain the method of pyrosequencing

Answer 36

A long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser.
These lengths are then degraded into single-stranded DNA (ssDNA). These are the template DNAs and they’re immobilised.
A sequencing primer is added and the DNA is then incubated with the enzymes DNA polymerase, luciferase and other stuff.
– When a complementary nucleotide is present it joins the chain
– The addition of a nucleotide to the chain releases energy
– The energy is used to activate a protein called luciferin
– Light released by luciferin is detected.
– If two identical nucleotides are added together then the intensity of the light emitted is doubled.

Question 37

How long is the human genome?

What is the problem with this?

Answer 37

Approximately 3.2 billion nucleotides long but even high throughput sequencing methods can only sequence a maximum of about 15,000 bases at a time.

Question 38

How do you sequence a whole genome?

Answer 38

Extract samples of DNA from cells. 
Cut DNA into sections of varying length.
Amplify the DNA (create many copies).
Sequence short sections of DNA.
Place sections in order by matching overlapping regions.

Question 39

What are the aims of the human genome project?

Answer 39

To work out the order or sequence of all the three billion base pairs in the human genome.
To identify all the genes.

Question 39

How can the knowledge of the human genome project seen as beneficial?

Answer 39

Improved genetic testing.
Location of genes that might be linked to increased chances of inheriting a disease.
New gene therapy.
New knowledge of how humans have evolved.
Personalised medicines.
Synthetic biology.

Question 41

What is bioinformatics?

What is it also known as?

Answer 41

A branch or biology that has grown out of DNA sequencing research that uses computers to store the huge amounts of data generated. Eg: sequences genomes. It would have been impossible to store and analyse these data prior to computers and microchips. Specialist software packages are specially designed for this purpose and can be used to compare genomes between species and between individuals of the same species.
Computational biology.

Question 42

Explain 5 ways in which genomes can be compared between species

Answer 42

Evolutionary relationships can be explored by considering the similarities in the genomes of two species. Beneficial genes are conserved by evolution. This has led to the reclassification of some organisms.
Identifying genes which have been altered give rise to differences between organisms e.g. FOXP2 gene is slightly different in humans as it has enabled speech.
The identification of genes common to most/all living things can give clues as to the relative importance of these genes to life.
Differences in gene interaction leading to different proteins being produced can be researched.
Medical research can be carried out, by comparing the genome of pathogenic and non‐pathogenic bacteria, to identify the genes responsible for causing disease.

Question 43

Explain 4 ways in which genomes can be compared between individuals

Answer 43

Epigenetics is the study of changes in organisms by the modification of gene expression rather than changes of the genetic code. Methylation of DNA can influence gene regulation, and thus mapping the methylation of a whole genomes can help our understanding of diseases like cancer do or do not develop in genetically similar individuals.
Investigate the relationship between which genotypes cause which phenotypes.
Early human migration can be mapped by comparing the genomes of humans from around the world.
Medical advances can be made by possibly producing drugs specific to an individuals genome, to maximise its effect.

Question 44

What has gene sequencing lead to the development of?

What is this?

Answer 44

Synthetic biology.
Designing and building useful biological devices and systems such as: producing medication, detecting and cleaning pollution.

Question 45

List and explain the 5 applications of synthetic biology

Answer 45

Biosensors - genetically engineered bioluminescent bacteria coat a microchip and glow if petroleum air pollutants are present.
Info storage - scientists can encode huge amounts of digital info onto a single strand of synthetic DNA.
Nanotechnology - produces materials eg: amyloid fibres for making biofilms for adhesion. Microbes will stick to these - they can be used to clean waste water.
Novel proteins - specially designed eg: haemoglobin that won’t bind to CO.
Production of medicines - engineering bacteria/fungi to produce the active ingredient of a drug otherwise hard to extract (often to extract).

Question 46

Explain why a mutated allele may cause a genetic disease

Answer 46

Mutated allele = wrong sequence of DNA bases.
Wrong sequences of DNA bases get transcribed into wrong mRNA strand.
Wrong mRNA strand translated into wrong sequence of amino acids
Wrong sequence of amino acids = wrong polypeptide = wrong shape
of protein = wrong/none functioning protein = symptoms of disease.

Question 47

Define gene therapy.
What is its basic principle?
What can it treat?
State and define the 2 types of gene therapy.

Answer 47

Treating genetic disorders using genetic technology.
The basic principle of gene therapy is to insert a functional allele of a particular gene into cells that contain only mutated and non-functioning alleles of that gene.
It could treat diabetes, SCID, parkinsons, cystic fibrosis etc.
Somatic cell gene therapy - gene therapy by inserting functional alleles into body cells.
Germ line gene therapy - gene therapy by inserting functional alleles into gametes or zygotes.

Question 48

Define what somatic cells are.
Define what specialised cells are.
Define augmentation.
Define what an ex vivo does.
Define what vectors are.
Define what killing cells are.

Answer 48

Normal body cells (not gametes).
Some gene on/off to produce/not produce specific proteins.
Adding a functional version of a gene so that the correct protein is made = relieves symptoms. Introducing new genes can be difficult (ex vivo or vectors).
Take cells out, modify then replace.
A virus or liposome to deliver desired allele to cells (not very effective).
Cancerous cells genetically engineered to produce antigens which help the body’s immune system recognise the cancer cells and be able to destroy them.

Question 49

What do patients with cystic fibrosis lack?

Answer 49

A functioning CFTR gene

Question 50

What are germ cells?

Explain germline gene therapy

Answer 50

Cells leading to the production of a new organism (gametes,
embryonic stem cells).
Genetically engineering gametes or zygote. All cells in the new organism will have the desired gene. May pass on desired gene to offspring.

Question 51

Germline gene therapy is done in animals but is illegal in humans. Why?

Answer 51

Could (unintentionally) introduce an genetic disease ‐/increase risk of cancer by gene being inserted into wrong location.
These are permanent changes to human DNA that can be passed on. This is considered unethical.
Could this lead to eugenics or ‘designer babies’?
Patient has no say in DNA being modified.

Question 52

Compare the 2 types of gene therapy. Include:
Ease of delivering genes,
Ability to pass genes on to offspring,
Ability to pass genes on to other cells/implications of this,
Ethics.

Answer 52

S: Harder to deliver genes. Has to be ex vivo or in vectors (which can be ineffective).
G: Delivering genes is easier as it is straight into germ cells.
S: Can’t pass on new genes to offsrping.
G: Can pass on new genes to offspring.
S: Specialised cells are treated and don’t divide. Can’t pass on genes to other cells. Need to repeat gene therapy regularly (as specialised cells are replaced).
G: No need torepeat therapy as every cell and hence every new cell will contain a copy of new genes.
S: Allowed in humans.
G: Not allowed in humans for ethical reasons ‐ danger of designer babies/eugenics.

Question 53

What are the advantages of obtaining insulin from genetically
engineered bacteria?

Answer 53

The old method of obtaining insulin was from pigs. Advantages over this method are:
Engineered insulin is cheaper (as foodstock is cheaper than that for pigs).
Much larger amount of product is more readily available as the rate of production is much faster.
There is less risk of infection than with pig insulin.
Human insulin is more effective for humans than pig insulin.
Avoids side effects/allergies/immune response that some people experience with pig insulin.
Ethically it is advantageous to use bacteria as there are no animal rights
issues associated with them as there are with pigs. This is also true for religious groups e.g. Jews who may not want pig insulin for religious
reasons.

Question 54

Outline the advantages and disadvantages of the genetic manipulation of animals (including humans). You must include:
Insect resistance in GM soya.
GM pathogens for research.
Pharming ‐ GM animals to make pharmaceuticals.
GM seed availability to poor farmers (patenting).

Answer 54

+ Pharming is getting GM animals to make pharmaceuticals. For example some proteins are too big for bacterial cells to make so GM animals are used to make them in their milk. Alpha antitrypsin can be made by GM goats to treat hereditary emphysema.
- The modification is not done with animal welfare in mind so animal could be uncomfortable/harmed.

Question 55

Outline the advantages and disadvantages of the genetic manipulation of plants. You must include:
Insect resistance in GM soya.
GM pathogens for research.
Pharming ‐ GM animals to make pharmaceuticals.
GM seed availability to poor farmers (patenting).

Answer 55

+ insect resistance in GM soya ‐ Bt gene inserted into soya plant DNA ‐ they produce the Bt toxin, killing insects which eat them and would reduce the crop yield. No need to use pesticide ‐ prevents possibly contaminating wild plants with insecticide/prevents human exposure when spraying insecticide.
Herbicide resistance in GM soya ‐ gene resistant to herbicide inserted to DNA ‐ crop can be sprayed, all weeds killed but crop not damaged ‐ can reduce the number of sprays needed.
- could crossbreed with wild plants and pass on insect/herbicide resistance to wild plants. This could reduce biodiversity. Wild plants may kill insects in the food chain. Super weeds resistance to herbicide could be made.
GM seeds are often patented which means that farmers may not legally be allowed to clone/fertilise/collect seeds of the plants they grow but would have to buy new seeds each year. Often it is poor farmers most in need of GM crops e.g. Golden Rice, drought resistant plants and this raises ethical questions about the availability of this technology.

Question 56

Outline the advantages and disadvantages of the genetic manipulation of microorganisms. You must include:
Insect resistance in GM soya.
GM pathogens for research.
Pharming ‐ GM animals to make pharmaceuticals.
GM seed availability to poor farmers (patenting).

Answer 56

+ GM pathogens are grown in labs for research into diseases, their metabolism and drugs to treat them.
Viruses are modified to be harmless to be vectors in gene therapy.
- GM pathogens could escape labs and cause epidemics.

Question 57

What can viruses be used as in gene therapy?

Explain how this is possible.

Answer 57

Vectors.
It’s a virus that usually affects humans is genetically modified so that it increases the functioning allele to be inserted into the patient, whilst at the same time being unable to cause a disease, it can enter the recipient cells, taking the value with it.

Question 58

What are the potential problems with using viruses as gene delivery agents?

Answer 58

Viruses, even though not virulent, may still provoke an immune or inflammatory response in the patient.
The patient may become immune to the virus, making subsequent deliveries difficult or impossible.
The virus may insert the allele into the patient genome in a location that disrupts a gene involved in regulating cell division, increasing the risk of cancer.
The virus may insert the allele into the patient’s genome in a location that disrupts the regulation of the expression of other genes.