Application of reproduction and genetics

Question 1

The human genome project:
when was the project proposed?
when did the project begin?
when was the first working draft published?
When was the more complete draft published?

Answer 1

• the project was proposed in 1985
• the project began in 1990
• the first working draft as published in 2000
• a more complete draft was published in 2003

Question 2

What were the aims of the human genome project?

Answer 2

• Identify all the genes in the human genomes and identify which chromosome each is on
• Determine the sequence of the 3 billion base pairs in human DNA and store this information in databases
• Improve tools for data analysis
• Transfer related technologies to the private sector, to develop medical innovation
• Address the ethical, legal and social issues that may arise from the project

Question 3

What were the main findings of the human genome project?

Answer 3

• Humans have about 20,500 genes, far fewer than anticipated
• There are more repeated segments of DNA than had previously been suspected
• Fewer than 7% of the families of proteins were specific to vertebrates, emphasising the close relationships between all living organisms

Question 4

Explain the process of Sanger sequencing (Chain termination)

• How do you make dideoxynucleotides

Answer 4

1. DNA was broken into single-stranded fragments of different lengths, up to 800 bases long
2. Complementary strands were synthesised but these were incomplete because the 4 nucleotide triphosphates (NTP) were altered. For each NTP, the 3’OH of the deoxyribose was removed, to make dideoxynucleotide i.e. lacking both the 2’OH and the 3’OH
3. When it was incorporated into newly synthesised strand, DNA polymerase would not be able to bind the next nucleotide and the chain could not lengthen i.e. the chain was terminated
4. This last nucleotide was marked with a radioactive isotope, an antigen or fluorescent marker, with a different one for each of the 4 nucleotides
5. From each original DNA fragment, a large number of complementary DNA strands of increasing length were produced. They were separated by gel electrophoresis, according to their size, and the terminal, marked nucleotide was identified
6. As all of the terminal nucleotides were known, in fragments of increasing size, the base sequence of the DNA fragment was known

Question 5

The 100K Genome Project:
When was the project launched?
Whose genomes were sequenced ?
Who was the project run by?

Answer 5

• The project was launched in 2012
• The project uses NGS to sequence 100,00 genomes from NHS patients with cancer or rare disease, and from members of their families
• It was run by genomics England, under the department of health

Question 6

Aims of the 100K Project:

Answer 6

• Create an ethical, transparent programme based on consent
• Set up a genomic service for the NHS to benefit patients
• Enable medical and scientific discovery
• Develop a UK genomics industry

Question 7

Moral and ethical concerns: Ownership of genetic information

Answer 7

Once base sequences are known, it must be clear who owns the information. If It is the property of the individual, then safeguards must be put in place that it is not misused e.g:

• If a person is identified as having a DNA sequence that might predispose them to heart disease, this information should not be used to set their insurance premiums or to deny them health and life insurance
• If a DNA sequence suggests a particular ancestry, this should not be a pretext for social discrimination
• No company should make financial profit from using a DNA sequence without permission

Question 8

Moral and ethical concerns: The identification of allele sequences

Answer 8

• A patients DNA can be scanned for mutated sequences that may be correlated with future health problems.
• Some people do not wish to have this knowledge about themselves. Some of the information may affect close relatives, it must be clear whether relatives have a right the information

Question 9

Moral and ethical concerns: Genetic screening and counselling

Answer 9

• If a family has a history of a genetic defect, family members can consult a genetic counsellor for advice on the risk for themselves or potentially for their children

Question 10

Moral and ethical concerns: Embryo screening

Answer 10

• Embryos made during IVF can be screened for the presence of alleles leading to conditions including cystic fibrosis
• A choice can then be made to implant the healthy embryo

Question 11

Moral and ethical concerns: Embryo screening for the purpose of ‘desirable’ characteristics

Answer 11

• potential for choosing alleles to ensure specific characteristics e.g appearance or sporting ability

Question 12

Moral and ethical concerns: parents may wish to have their children screened

Answer 12

• to know if they carry sequences that predispose them to adult diseases so a decision must be made to whether or when to tell the child the results of the tests
• Suggests that the childs DNA is the property of the parent

Question 13

Moral and ethical concerns: storage and security

Answer 13

a concern because of the potential for computer storage to be hacked

Question 14

Why examine genomes?

Answer 14

• allows inferences to be drawn concerning evolutionary relationships
• these provide a true phylogenetic classification
• comparisons indicate to conservation scientists which species need particular conservation

Question 15

Killing the vector: Anopheles gambiae

-resistance

Answer 15

• Insecticides are used in indoor sprays to kill mosquitos in buildings
• Pyrethroid resistance is a particular problem, as that is the only insecticide recommended for use with the nets under which people sleep

Question 16

DNA sequencing of Anopheles Gambiae

Answer 16

• performed in 2002, the sequence is used to try to develop chemicals that can prevent the mosquito from transmitting malaria, by making it susceptible to insecticides

Question 17

How was a genetically modified mosquito produced in 2015?

Answer 17

• Produced using gene-editing technology
• Mosquito eggs modified with the addition of a gene that would allow them to synthesise an antibody against Plasmodium
• Then if the mosquito acquired Plasmodium when taking blood from an infected person, the plasmodium would not survive in the mosquito
• The mosquito would not then spread the infection when biting people in the future

Question 18

Killing the parasite: Plasmodium falciparum

Answer 18

Drugs to kill P.F have been used since the early 17the century when extracts from Cinchona bark were first used

• QUININE: Disrupts P.F’s the digestion of haemoglobin in the red blood cells. A toxic derivative of haemoglobin accumulates and kills the Plasmodium
• CHLOROQUINE: disrupts the digestion of haemoglobin in P.F’s food vacuole. Expels the drug from its food vacuole 50 x faster than normal- not enough time for drug to have an effect (resistance)
• ATOVAQUONE: kills P.F by acting on the electron transport chain in its mitochondria. Resistance to develops rapidly caused by single point mutation in the gene for cytochrome b
• ARTIMESININ: acts on P.F’s red blood cells

Question 19

what is a persons DNA profile?

How much difference is there between individuals?

Answer 19

their genetic fingerprint

0.1% difference that makes an individuals genetic fingerprint unique

Question 20

Why is a genetic fingerprint not the same as a DNA sequence

Answer 20

it only represents non-coding portions of DNA

Question 21

DNA fingerprinting relies on two techniques

Answer 21

1. The polymerase chain reaction to make large numbers of copies of DNA fragments
2. Gel electrophoresis, to separate the DNA fragments based on their size

Question 22

how much human DNA codes for proteins?

Answer 22

less than 2% of human DNA codes for proteins

Question 23

what is the difference between introns and exons

Answer 23

introns do not code for proteins and they have sequences of nucleotides in which up to 13 bases repeat up to several hundred times exons do code for proteins

Question 24

what makes genetic fingerprints unique?

Answer 24

the number of repeats within a particular STR is different individuals

Question 25

What is the polymerase chain reaction (PCR)

Answer 25

PCR is semi-conservative replication of DNA in a test tube. It greatly amplifies the DNA i.e. makes millions of copies and it works rapidly

Question 26

The DNA sample is dissolved in a buffer and mixed with:

Answer 26

• Taq polymerase. This is DNA polymerase from the bacterium Thermus aquaticus which lives in hot springs and hydrothermal vents. Taq polymerase has an optimum temperature of about 80 degrees
• Active for 9 minutes before denaturing
• Nucleotides containing the four DNA bases
• short single stranded pieces of DNA between 6 and 25 bases long, called primers. They are complementary to the start of the DNA strand and bind to it, signalling taq polymerase to start replication

Question 27

what does PCR depend on?

Answer 27

rapid temperature change, which happens in a device called a ‘thermocycler’

Question 28

Explain the stages of PCR

Answer 28

1. The original ‘target’ DNA is heated to 95 degrees, separating it into two strands
2. The solution is cooled to 55 degrees, which is cool enough for the primers to anneal to the complementary strand by adding complementary nucleotides and catalysing the formation of phophodiester bonds in the sugar-phosphate backbone.
3. This is the elongation or extension phase. For each initial fragment of double-stranded DNA, two identical double strands are produced
4. The sequence is repeated many times

Question 29

How were copies of genes made before PCR was invented?

Answer 29

by inserting them into replicating microorganisms

Question 30

Limitations of PCR: Contamination

Answer 30

Any DNA that enters the system by accident can be amplified. The contaminating DNA may be air-borne, come from the experiment or from contaminated reagents. But most contamination comes from previous PCR reactions using the same apparatus

Question 31

Limitations of PCR: Error rate

Answer 31

All DNA polymerases sometimes insert a nucleotide containing the wrong base. They usually proofread and correct their errors, but taq polymerase cannot do this. It makes an error about 1 in every 9000 nucleotides. After 30 cycles of PCR, the error rate becomes 1 per 300 nucleotides, because each cycle copies and multiplies the previous errors, so they accumulates

Question 32

Limitations of PCR: DNA fragment size

Answer 32

PCR is most efficient for making DNA about 1000-3000 base pairs long because taq polymerase cannot correct its errors. If a lower temperature, higher pH and proofreading polymerase in addition to taq polymerase are used, a length of 40,000 base pairs can be generated. But many genes, including human genes, are much longer than this

Question 33

Limitations of PCR: Sensitivity to inhibitors

Answer 33

molecules in the sample may act as inhibitors and PCR is very sensitive to them e.g.:

• Phenolics, especically in plant material
• Humic acids in in archaeological specimens
• Haem breakdown products, which bind with Mg2+ needed for the polymerase to function

Question 34

Limitations of PCR: Limits to amplification

Answer 34

At the start of PCR, the number of DNA molecules made increases exponentially. After about 20 cycles, it slows down: the increase becomes linear and then plateaus because:

• reagent concentrations become limiting
• the enzyme denatures after repeated heating
• DNA in high concentrations causes the single-stranded molecules to base pair with each other with the primers

Question 35

Explain the process of gel electrophoresis

Answer 35

1. DNA is extracted from biological material and cut into thousands of fragments of varying lengths, using restriction endonucleases
2. The DNA fragments are separated by length with gel electrophoresis, on an agarose gel. Agarose is a polysaccharide extracted from seaweed and makes a gel with pores, through which small molecules can move:
   - DNA samples are loaded into wells of the gel
   - A voltage is applied across the gel. The phosphate groups of the DNA backbone have a negative charge so the fragments are attracted to the anode. Smaller fragments move more easily through the pores and so they migrate through the gel faster than the larger fragments
   - If fragments of known length are separated on the same gel at the same time, making a ‘DNA ladder’ the lengths of the fragments under test can be estimated
3. The electrophoresis trough is covered with a nylon membrane, which touches the gel and picks up the DNA fragments. This process is called Southern blotting
4. Radioactive or, more commonly luminescent DNA probes that contain sequence complementary to the STRs attach by base pairing to specific of the fragments. Any unbound probes are washed off
5. A film that is sensitive to X-rays or the wavelengths emitted by the luminescent probe is placed over the Southern blot overnight
6. The film is exposed and the autograph reveals a banding pattern in which the dark bands show the position of the probe, and therefore the repeated sequences. This pattern is the genetic fingerprint

Question 36

DNA profiling has been used in many situations, including testing for: Paternity

Answer 36

• The DNA from white blood cells is used to construct DNA profiles.
• The bands in a child’s profile are compared with the mothers
• Any bands that they share were inherited from her
• The remaining bands in the child’s profile must have been inherited from the father
• If they do not match, there is a high probability that he is the father, but this technique cannot provide absolute proof

Question 37

DNA profiling has been used in many situations, including testing for: Twins

Answer 37

• Monozygotic (identical) have identical banding patterns in their DNA profile whereas dizygotic (fraternal) twins or not
• At birth, twins may look very similar so this is a useful tool for distinguishing which type of twin they are

Question 38

DNA profiling has been used in many situations, including testing for: Siblings

Answer 38

• People who have been adopted may wish to confirm that alleged biological siblings are in fact their blood relatives
• If they are, their DNA profiles will show many similarities, as, on average, half their genes will be identical

Question 39

DNA profiling has been used in many situations, including testing for: Immigration

Answer 39

• Some visa applications depend on proof of relatedness

Question 40

DNA profiling has been used in many situations, including testing for: Forensics

Answer 40

• Use to identify and rule out suspects in criminal cases

Question 41

DNA profiling has been used in many situations, including testing for: Phylogenetic studies

Answer 41

• Profiles of members of different taxa can be compared to determine whether they have been classified suitably and to determine how closely they are genetically related

Question 42

What are the pros of DNA profiling?

Answer 42

• It does not require an invasive method to obtain a biological sample, as mouth swabs, urine or hair can be used to obtain DNA, rather than blood samples
• The technique can be used on samples that would be too small for blood testing
• It has reversed wrongful convictions when used with other forensic tools and evidence
• DNA profiling can rule out non-matches of DNA samples to exonerate people who have been falsely accused
• Efforts are being made to store genetic material from people around the world, before isolated groups are intermixed and lost

Question 43

What are the cons of DNA profiling?

Answer 43

• Some people consider that any request for a DNA sample is a violation of an individuals right to privacy and their civil liberties
• DNA profiles are held in computer databases, which are vulnerable to misuse and hacking. Individuals may suffer loss of privacy; information taken ostensibly of an apparent match being correct may rise to one in one hundred million
• Access to and use of data must be carefully regulated, as with DNA sequencing, Health insurers could conceivably use it to deny coverage or claims; prospective employers could avoid hiring those who have certain genetic traits or risks for certain diseases, private information, such as gender reassignment, could be made public without consent

Question 44

DNA profiling may produce wrongful convictions if:

Answer 44

• It is used inappropriately to influence juries and judges, especially if they lack an understanding of the significance of the results
• Errors may occur in the procedure
• People conducting the tests may not be trustworthy
• DNA evidence is planted at a crime scene

Question 45

What is genetic engineering?

Answer 45

Genetic engineering allows genes to manipulated, altered and transferred from one organism or species to another, making a genetically modified (GM) organism.

Question 46

Applications of genetic engineering include the transfer of genes or gene fragments into:

Answer 46

• Bacteria so that they can make useful products, such as insulin
• Plants and animals, so that they acquire new characteristics, for example resistance to disease
• Humans, to reduce the effects of genetic diseases, such as Duchenne muscular dystrophy