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Module 6- Genetics and Ecosystems > 6.3- Manipulating genomes > Flashcards

6.3- Manipulating genomes Flashcards

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1
Q

Define DNA sequencing

A

A technique that allows genes to be isolated and read

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2
Q

outline the general steps of Sangers DNA sequencing

A

1) cloning the DNA
2) DNA split into single strands
3) DNA sequenced

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3
Q

What is electrophoresis

A

A technique used to separate different sized fragments of DNA or proteins- separates for identification and analysis- can separate fragments that differ by only 1 base pair

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4
Q

Outline the basis of electrophoresis

A
  • uses agarose gel plate covered by a buffer solution
  • electrodes placed in each end of tank- when connected to power supply, an electric current can pass through gel
  • DNA has overall negative charge due to phosphate groups- means they migrate towards anode (positive electrode)
  • fragments of DNA all have similar surface charge regardless of their size
  • The DNA fragments move through the gel at different speeds- smaller travel faster so in a set period they travel further
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5
Q

Describe the process of electrophoresis

A

1) DNA samples digested with restriction enzymes to cut them at specific specific recognition sites into fragments at 35-50 degress (may take up top an hour)
2) tank is set up during this- arabise gel made and poured into central regions- combs placed at one end whilst gel is setting- once set an be removed
3) Buffer added to surface if set gel so that gel is covered
4) Loading dye is added to tubes containing the digested DNA
5) The digested DNA and the loaded due is added to the wells in the electrophoresis gel- pipetted used and geld in buffer solution above wells (not in as may pierce the bottom)- loading dye is dense and carries the DNA into the the well
6) Once all of the wells have been loaded with the different DNA samples- electrodes are put into place and connected to 18V battery- left to run for 6-8 hours (or could use higher voltage for less term if current is less- 5mA as electric shock possible)
7) The DNA fragments move through the gel at different speeds- smaller travel faster so in a set period they travel further
8) at the end of the period, the buffer solution is poured away and a dye is added to the gel- this dye adheres to the DNA and stains the fragments

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6
Q

What is another use for electrophoresis (except for separating fragments of DNA)

A

Separating proteins

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7
Q

Describe the use of electrophoresis for separating proteins

A
  • principle same as for separating DNA fragments but often carried out in the presence of a charged detergent such as sodium dodecyl sulphate (SDS) which equalises the surface charge in the molecules and allows the proteins to separate as they move through the gel according to heir molecular mass
  • in some cases the proteins can be separated according to mass and them, without SDS, according to their surface charge
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8
Q

Describe applications of using electrophoresis to sequence proteins

A

Can be used to analysed the types of haemoglobin proteins for diagnosis of conditions such as:
- sickle cell anaemia- patient has haemoglobin S and not normal haemoglobin A
- aplastic anaemia, thalassaemia and leukaemia- patients have higher than normal amounts of fatal haemoglobin (haemoglobin F) and lower than normal amounts of haemoglobin A

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9
Q

What does PCR stand for

A

Polymerase chain reaction

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10
Q

Define the polymerase chain reaction

A
  • a biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
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11
Q

Outline the reason for PCR

A
  • used in forensic analysis
  • DNA profiling can obtain results from as few as 5 cells but criminal may have innocent people DNA on their hands from touching frequently touched surface (as all leave skin cells behind)
  • Mullis developed PCD to amplify DNA, enabling it to be analysed
  • thus became part of protocols for analysis of DNA for genetic diseases and forensic DNA analysis
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12
Q

What does PCR rely on

A
  • DNA is made of 2 anti-parallel backbone strands
  • each strand of DNA has a 5’ and a 3’ end
  • DNA grows only from the 3’ end
  • base pairs pair up according to complimentary base pairing rules (A-T, C-G)
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13
Q

How does PCR differ from DNA replication

A
  • only short sequences (of up to 10,000 base pairs) can be replicated, not entire chromosomes
  • requires addition of primer molecules to make process start
  • cycle of heating and cooling needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated
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14
Q

Outline the principles of the PCR

A
  • cyclic reaction
  • the amount of DNA increases exponentially- 1-2-4-8-16-32-64-128 etc
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15
Q

How do you calculate the number of new strands from the PCR

A

2 to the power of how many replications there were

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16
Q

PCR diagram

A
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17
Q

Why was the PCR time-consuming at first, what then changed

A
  • DNA was heated to denature it and then cooled to around 35 to anneal the primers and allow the DNA polymerase to work
  • later, DNA polymerase was obtained thermophilic bacterium tehrmophilus aquaticus- called Taq polymerase and is stable at high temperatures- optimum temperature of around 72
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18
Q

Describe the steps of the PCR

A
  1. Sample of DNA mixed with DNA nucleotides, primers, magnesium ions enzyme Taq DNA polymerase
  2. mixture heated to 94-96 °C- breaks the hydrogen bonds between complementary nucleotide base pairs and thus denature the double-stranded DNA into two single strands
  3. Mixture cooled to 68 °C, so that the primers can anneal (bind by hydrogen bonding) to one end of each single strand of DNA- gives a small section of double-stranded DNA at the end of each single-stranded molecule
  4. The Taq DNA polymerase enzyme molecules can now bind to the end where there is double-stranded DNA
  5. Temperature is raised to 72 °C, which keeps the DNA as single strands
  6. The Tag 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 5’ to 3’ direction.
  7. When the Tag DNA polymerase reaches the other end of the DNA molecule, then a new double strand of DNA has been generated
  8. The whole process begins again and is repeated for many cycles
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19
Q

Describe applications of PCR

A
  • tissue typing- donor and recipient tissues can be typed prior to translation to reduce the risk of rejection of the transplant
  • direction of oncogenes- if the type of mutation involved in a specific patient cancer is found, medication may be tailored to that patient
  • detecting mutations- a sample of DNA is analysed for the presence of a mutation that leads to a genetic disease- parents can be tested o see of they carry recessive allele, fatal cells may be obtained from mothers bloodstream for prenatal eugenic screening, during IV one cell from 8-celll embryo may be used to analyse the fatal DNA before implantation
  • identifying viral infections- sensitive PCR tests ca detect small quantities of viral genome amongst host cells DNA- can be used to verify GIV or hepatitis C
  • monitoring spread of infectious diseases- spread of pathogens through. population of wild/domestic animals pr from animals to humans can be monitored and emergence of more virulent sub0types can be detected
  • forensic science- small quantities of DNA can be amplified for DNA profiling to identify criminals or ascertain parentage
  • research- amplifying DNA from extinct sources e.g. neanderthal/woolly mammoth bones for analysis and sequencing. In extant organisms, tissues or cels can be analysed to find out who genes are switched on/off
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20
Q

Why are primers needed in PCR

A

DNA polymerase cannot bind to single stranded DNA

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21
Q

describe the cloning of DNA in Sangers DNA sequencing approach

A
  • the gene being sequenced is isolated from a bacterium (cut) using restriction enzymes
  • the DNA then inserted into a bacterial plasmid (the vector) and then into an escherichia coil bacterium host that, when cultured, divided many times
  • enables the plasmid with the DNA insert to be copied many times
  • each new bacterium contained a copy of the candidate gene- these length of DNA now isolated using plasmid preparation techniques
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22
Q

What is another term for Sanger’s DNA sequencing approach

A

Chain termination

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23
Q

Describe how strands of DNA were split into single strands for Sangers DNA sequencing

A
  • heated to around 94-96- breaks the hydrogen bonds between the complimentary nucleotide base pairs- denatures the double-stranded DNA into 2 single strand of DNA
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24
Q

Describe how DNA is sequenced using Sanger’s approach

A
  • uses copies of a single strand of DNA as a template for 4 experiments
  • 4 separate dishes
  • each of the 4 dishes contains a solution with the 4 bases (AT,T,C,G)- with a modified version of one of the bases in each dish
  • this modified base was modified in such a way that once incorporated to the complimentary strand of DNA, no more bases could be asses, and was labelled with a radioactive isotope
  • as the reaction progressed thousands of DNA fragments of varying lengths generated
  • DNA fragments passed through Gell by electrophoresis- sorted by length
  • nucleotide base at the end of each fragment read according to radioactive label
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25
Q

Describe an example of interpreting results from Sangers DNA sequencing

A
  • If the first one-base fragments had thymine at the end, then the first base in the sequence is T
    *If the two-base fragments have cytosine at the end, then the sequence is TC.
  • If the three-base fragment ends with guanine, then the base sequence is TCG.
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26
Q

Sangers DNA sequencing diagram

A
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27
Q

Sangers DNA sequencing results interpretation diagram

A
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28
Q

Describe the practical use of Sangers DNA sequencing

A
  • method was efficient and safe- Sanger used it to sequence genome of a phage virus (virus that infects bacteria)- first DNA based organism to have genome sequenced
  • also sequenced human mitochondrial genome
  • later scientists sequenced 170 kilo base genome of Epstein-Barr virus
  • however, was time consuming and therefore costly as have too count off the bases one by one from the bands in a piece of gel
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29
Q

Describe the development of Sangers technique to DNA sequencing

A
  • 1986- first automated DNA sequencing machine developed at California institute of technology- based on Sangers method
  • fluorescent dyes instead of radioactivity used to label terminal bases
  • the dyes glowed when scanned with a laser bam, and the light signature was identified by computer
  • involves reading autoradiograms
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30
Q

Name a more modern technique of DNA sequencing to Sangers approach

A

pyrosequencing- developed in 1996

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31
Q

Briefly outline pyrosequencing

A

involves synthesising a single strand of DNA, complementary to the strand to be sequenced one base at a time, while detaching which base was added at each step by light emission

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32
Q

Describe the process of pyrosequencing

A
  1. A long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser
  2. These lengths are then degraded into single-stranded DNA (ssDNA)- template DNAs that are immobilised
  3. A sequencing primer is added
  4. DNA is then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apvrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin
  5. One activated nucleotide (a nucleotide with two extra phosphoryl groups), such as TTP (thymine triphosphate), is incorporated into a complementary strand of DNA using the strand to be sequenced as a template- only one of the 4 possible activated nucleotides is added at any one time
  6. As this happens, the two extra phosphoryls are released as pyrophosphate (PP). In the presence of APS, the enzyme ATP sulfurylase converts the pyrophosphate to ATP. In the presence of this ATP, the enzyme luciferase converts luciferin to oxyluciferin. This conversion generates visible light which can be detected by a camera
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33
Q

What is the difference between pyrosequencing and Sangers method

A

It uses sequencing by synthesis rather than by chain termination

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34
Q

Describe interpretation of pyrosequencing

A

the amount of light generated is proportional to the amount of ATP available and, therefore, indicates how many of the same type of activated nucleotide were incorporated adjacently into the complementary DNA strand

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35
Q

What is an activated nucleotide (pyrosequencing)

A

a nucleotide with two extra phosphoryl groups

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36
Q

What happens at the end of pyrosequencig

A
  • incorporated activated nucleotides are degraded by apyrase and the reaction starts again with another nucleotide
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37
Q

Describe the speed/timing of pyrosequencing

A
  • on million reactions occur simultaneous
  • means. a10-hour run generates 400 million bases of sequencing information
  • software packages assemble these sequences into longer sequences
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38
Q

Pyrosequencing diagram

A
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39
Q

Name a branch of biology involved in DNA sequencing

A

Bioinformatics

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40
Q

Describe bioinformatics

A
  • has grown out of pyrosequnecing research, to store huge amounts of data generated
  • would have been impossible to store and analyse these data prior to computers and microchips
  • software packages are specially designed for this purpose
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41
Q

Reading pyrosequencing results graph

A
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42
Q

Name applications of gene sequencing

A
  • the human genome project
  • genome-wide comparisons between individuals and species, including evolutionary relationships
  • predicting the amino-acid sequence of proteins
  • synthetic biology
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43
Q

Outline the human genome project

A
  • scientists predicted that human genome would contain around 100,000 genes- launched the HGP in 1990 and genome was finished by 2003
  • found genome contained only around 240000 genes- not many more than mouse genome
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44
Q

What is included in the genome of eukaryotes

A

the complete DNA sequence of an organisms genome- genetic material the chromosomes, mitochondria and chloroplasts if plant/algae

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45
Q

Where are sequenced genomes stored

A

gene Banks

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46
Q

Describe use of gene sequencing for comparisons between species

A
  • when human genome was compared with those of other organisms, found that few human genes are unique to us- most have counterparts in other organisms e.f share over 99% of our genes with chimpanzees
  • verifies that genes that work well are conserved by evolution e.g. why pigs/humans have similar genes for insulin (why pig insulin could be used to treated diabetes prior to GM bacteria)
  • sometimes as evolution progresses, some genes are co-opted to perform new tasks- e.g. tiny changes to human FOXp2 gene (also found in mice and chimpanzees) means that in humans this gene allows speech
  • many of the differences between organisms are not because the organisms have totally different genes, but because some of their shared genes have been altered and now work in subtly different ways
  • some changes to DNA that do not code directly for proteins have also altered the expression of the genomes- regulatory and coding genes interact in such ways that, without increasing the number of genes, the numbers of proteins made may be increased
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47
Q

Outline the development of DNA profiling

A
  • Alec jeffery- located tandem repeat sequences of DNA- 1978
  • realised a persons DNA profile could confirm or refute paternity and maternity
  • first method involved restriction fragment length polymorphism analysis- but this is laborious and is no longer used0 now use STRs
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48
Q

Describe the process of DNA profiling

A

1) DNA obtained from the individual- either by mouth seal, saliva on toothbrush, blood, hair, or in case of ancient remains from bone
2) DNA digested with restriction enzymes- cut DNA at specific recognition sites- these are the STR;s so the fragments will vary in size across people
3) The fragments are separated by gel electrophoresis and stained- larger fragments travel the shortest distance in the gel
4) A banding pattern can be seen
5) The DNA to which the individuals is being compared is treated with he same restriction enzymes and subjected to electrophoresis
6) The banding patterns of the DNA samples can be seen

49
Q

Describe the relevance of STR’s to DNA profiling

A
  • called short tandem repeat dequences
  • highly variable short repeating lengths of DNA
  • around 13 in the genomes
  • have different numbers of a repeating sequences of bases
  • exact number of STRs vary from person to person
  • each STR is polymorphic, but the number, but number number of alleles in the gene pool for each one is small
  • each STR is present in between 5% and 20% of individuals, but the chances of sharing STR sequences at all the loci is 1 x 10^18- larger than number of people on earth
  • however estimated 12 million identical twins
50
Q

Name applications of DNA profiling

A
  • Forensic science
  • maternity and paternity dispute
  • analysis of disease
51
Q

Describe use of DNA profiling in forensic science

A
  • transformed forensic sincere- brought about convictions, established innocence of many aspects and those wrongly convicted
  • e.g. used to identify Nazi war criminals hiding in South America, identify remains of Richard III in Leister, identify victims body parts after air crashed or terrorist attacks, or identify unidentified soldiers from war
52
Q

Describe use of DNA profiling in maternity and paternity disputes

A
  • half of every Childs genetic information comes from the mother and half from he father
  • means half the STR’s come from each
  • comparing DNA profiles of mother, father, and child can therefore establish maternity and/or paternity
53
Q

Describe use of DNA profiling in analysis of diseases

A
  • protein electrophoresis decent 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 can be detected by electrophoresis
54
Q

What are DNA probes

A
  • a short (50-80 nucleotides) single stranded length of DNA tat is complimentary too a section of the DNA being investigated
  • it is labelled
55
Q

Describe how DNA probes can be labelled

A
  • a radioactive marker, usually with 32P in one of the phosphate groups in the probe strand, Once a probe has annealed (bound) by complimentary base ring, to the piece of DNA, it can be revealed by exposure to photographic film
  • a fluorescent marker emits a colour on exposure to UV light- may also be used I automated DNA sequencing
56
Q

Describe applications of using a probe to locate specific DNA sequences

A
  • locating a specific gene needed for use in genetic engineering
  • to identify the same gene in a variety of different genomes 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
57
Q

Describe microarrays

A
  • a number of different probes on a fixed substance- a DNA microarray
  • applying the DNA under investigation to the surface can reveal the presence of mutated allies that match the fixed probes, as the sample DNA will anneal to any complimentary fixed probes
  • the sample DNA must be broken into smaller fragments, and it may ansi be applied using the PCR
  • a DNA microarray can be made with fixed robes, 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 both bind to a particular probe, the scan revealed fluorescence of both colour, including the presence of the particular sequence in the test DNA
58
Q

Describe the use of gene sequencing to the study of evolutionary relationships

A
  • comparing genomes of organisms thought to be closely related species has helped confirm their evolutionary relationships or has led to new knowledge about relationships, and in some cases has led to certain organisms being re-classified
  • e.g. the DNA from bones and teeth of some extinct animals can be amplified and sequenced, so that the animals evolutionary history can be verified
  • e.g. samples of extinct cave bear (Urus spelaeus) genomes were sequenced using high-throughput techniques, and he sequence data obtained was compared to those of dogs- found dogs and bears diverged around 50 million years ago and share 92% of their genome
59
Q

Describe the use of gene sequencing to the study of variation between individuals

A
  • all humans genetically similar- except for rare cases where a gene has been lost by deletion of part of a chromosome, we all share the same genes but we have different alleles
  • about 0.1% of our DNA is not shared with very small (as genome has 3 billion base pairs, there are 3 million places on the DNA lengths where our DNA sequences can differ due too random mutations such as substitution
  • the places on the DNA where these substitutions occur are called single nucleotide polymorphisms (SNPs)- some have no effect on the protein, some can alter a protein or alter the way a piece of RNA regulates the expression of another gene
  • methylation of certain chemical groups in DNA plays a major role in regulating gene expression in eukaryotic cells- methods to map this methylation of whole human genomes can help researchers to understand the development of certain diseases, for example certain types of cancer, and why they may or may not develop in genetically similar individuals- epigentcis
60
Q

Describe the use of gene sequencing to predict the amino acid sequence of proteins

A
  • determining the sequence of amino acids within a protein is laborious and time consuming
  • however, if researchers have the organisms genome sequenced and know which gene codes for.a specific protein, by using knowledge of which base triplets code for which amino acids, they can determine the primary structure of proteins
  • the researchers need to know which part of the gene codes for exons and which codes for introns
61
Q

Describe the use of gene sequencing in synthetic biology

A
  • interdisciplinary science concerned with designing and building useful devices and systems
  • it encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics
  • its ultimate goals may be to build engineered biological systems that store and process information, provide food, maintain human health and enhance the environments
  • the sequences of DNA found by analysing genomes provide potential building block =s for synthetic biologists to build devices
62
Q

Describe applications of synthetic biology

A

Information storage:
- Scientists can encode vast amounts of digital information onto a single strand of synthetic
DNA e.g. one project has encoded the complete works of Shakespeare onto a strand of synthetic DNA

Production of medicines:
- Escherichia coli and yeast have both been genetically engineered to produce the precursor of a good antimalarial drug, artemisinin, previously only available by extracting it from certain parts of the Artemisia plants at particular times in the plant’s life cycle

Novel proteins:
- Designed proteins have been produced, for example one that is similar to haemoglobin and binds to oxygen, but not to carbon monoxide

Biosensors:
- Modified bioluminescent bacteria, placed on a coating of a microchip, glow if air is polluted with petroleum pollutants

Nanotechnology:
- Material can be produced for nanotechnology
- e.g. amyloid fibres for making biofilms - for functions such as adhesion.

63
Q

Outline bioethics

A
  • synthetic biology raises issues of ethics and biosecurity
  • extensive regulations already in place due to 30-40 years of using genetically-modified organisms
  • many advisory panels and many scientific papers written on how to manage the risks
  • synthetic biology not about making synthetic life forms from scratch, but about a potential for new systems with regards and associated risks to be managed
64
Q

Describe the development of genetic manipulation

A
  • Humans have been genetically modifying plants for thousands of years- all the crops grown around the world have genomes vastly different from those of their wild relatives, as a result of human intervention and selective breeding
  • Agriculture has changed the face of the landscape and produced domesticated breeds of animals and plants
  • However, selective breeding relies on a rather ‘hit or miss’ technique and may produce unexpected results
  • In the 1970s, the techniques of recombinant DNA technology allowed scientists to splice new genes into plant genomes- gave a more exact way of transforming crop plants, compared with inducing mutations or crossing different strains
  • Now specific genes conferring desirable traits can be excised from one organism and inserted into another, using a vector or a ‘gene gun’
65
Q

Briefly outline ethical issues of genetic manipulation

A
  • Some people are concerned about potential hazards and risks associated with genetic modification (GM)
  • However, the potential benefits have to be recognised and weighed against the potential hazards
  • Anti-GM campaigners want the technology abolished, which would also abolish any choice and the opportunity to exploit potential benefits
66
Q

Name genetically modified organisms which have potential hazards

A
  • microorganisms e.g. e.coli
  • plants e.g. Bt tobacco and Bt maize
  • soya beans (1st generation)
  • golden rice (2nd generation)
  • plantains (type of banana)
  • crop plants resistant to stress
  • pathogens
  • mice
  • pharmaceutical proteins
  • silk
67
Q

Describe potential benefits of genetically manipulating microorganisms e.g. e.coli

A
  • GM microorganisms can make human insulin to treat all diabetics (this was not possible using pig insulin), and human growth hormone to treat children with pituitary dwarfism
68
Q

Describe potential hazards of genetically manipulating microorganisms e.g. e.coli

A
  • Microorganisms could escape into the wild and transfer marker genes for antibiotic resistance to other bacteria
    However, the GM bacteria are also modified so they cannot synthesise an essential nutrient and therefore cannot live outside the lab
69
Q

Describe potential benefits of genetically manipulating plants e.g. Bt tobacco and Bt maize

A
  • In 1985, tobacco plants were genetically modified to produce the toxin normally produced by a bacterium, Bacillus thuringiensis- this toxin, Bt, had been used by organic farmers as a pesticide, as it is toxic to insects
  • Because the bacterial gene, Bt, was inserted into some crop plants, the GM plants produced the toxin, eliminating the need to spray it around the environment possibly contaminating other organisms
70
Q

Describe potential hazards of genetically manipulating plants e.g. Bt tobacco and Bt maize

A
  • Bt is toxic to monarch butterflies
    However, these butterflies do not take nectar from tobacco plants or maize plants in the wild; they feed on milkweed- despite many thousands of hectares of land in the USA being planted with Bt crops, the monarch butterfly has continued to thrive
71
Q

Describe potential benefits of genetically manipulating soya beans

A
  • example of first generation GM plant- main advantage appeared to e to production company making the pesticide
  • GM soya beans, resistant to a herbicide (Round-Up Ready™), were produced, so that weeds competing with the soya plants could be killed with the herbicide.
72
Q

Describe potential hazards of genetically manipulating soya beans

A
  • potential for the gene for herbicide resistance to pass into weeds, producing ‘superweeds’
    However, this does not appear to have happened to date.
73
Q

Describe potential benefits of genetically manipulating Golden rice

A
  • example of a second generation GM crop bred to be nutritionally enhanced
  • About 500000 children each year in India go blind, and some of them die, through lack of beta carotene, the precursor to vitamin A
  • Golden Rice™ (see Figure 2) was genetically modified to contain a gene from daffodils, so that beta carotene would be present in the rice grains- rice is the staple food in this region
74
Q

Describe potential hazards of genetically manipulating Golden rice

A
  • Some were concerned that farmers would have to buy the seed every year
    However, the company that developed this rice has offered free licences to farmers so they can keep and replant rice seeds
75
Q

Describe potential benefits of genetically manipulating plantains

A
  • type of banana that is a stable food in Kenya
  • A local biotechnology company in Kenya, Africa Harvest, is producing plantains that are nutritionally enhanced to contain more zinc
  • In areas where people eat very little meat, they may be deficient in zinc- an important enzyme cofactor and essential for regulating insulin secretion
76
Q

Describe potential hazards of genetically manipulating plantains

A
  • Public fear- some people fear eating food that contains foreign DNA and worry that the inserted genes will somehow be expressed in us
    However, all the food we eat contains genes and we digest the DNA with specific enzymes, nucleases and nucleotidases
77
Q

Describe potential benefits of genetically manipulating crop plants resistant to pests

A
  • The biotechnology company Africa Harvest is producing crops that are resistant to pests, so that when farmers sow these seeds, they do not need to use pesticides
  • better for the environment
  • good for farmers as every year in Africa, about 2000 people die through exposure to pesticides while applying them
78
Q

Describe potential hazards of genetically manipulating crop plants resistant to pests

A
  • There were concerns that local farmers might not want the GM seed and would not have the choice to buy non-GM seed
    However, Monsanto also sells non-GM seeds, but many farmers see the benefits of the GM seeds and do not want to be exposed to pesticides
79
Q

Describe potential benefits of genetically manipulating pathogens

A
  • Viruses, genetically modified to have no virulence, can be used to make vaccines, as they still have the antigens on their surfaces- reduces the chance of a vaccine making the recipient ill
  • Modified viruses can also be used as vectors in gene therapy
80
Q

Describe potential hazards of genetically manipulating pathogens

A
  • There have been some problems with the use of viruses in gene therapy, as the allele may be inserted into the genome in a way that increases the risk of cancer or interferes with gene regulation
81
Q

Describe potential benefits of genetically manipulating mice

A
  • Since 1974, millions of GM mice have been bred for medical research and used to develop therapies for breast and prostate cancer
  • Other types of mice have had certain genes knocked out, so that researchers can find out the function of those genes
82
Q

Describe potential hazards of genetically manipulating mice

A
  • Some people object to the use of animals for medical and pharmaceutical testing
    However, in the UK, strict regulations govern the welfare of animals used in this way and many of us have benefitted from medical protocols developed using animals
83
Q

Describe potential benefits of genetically manipulating pharmaceutical proteins

A
  • Genes for human pharmaceutical proteins, such as alpha antitrypsin to treat hereditary emphysema, can be inserted into goats or sheep, and the human protein they express into their milk is harvested
  • Transgenic mammals were used because this protein is too large for a bacterial cell to synthesise.
84
Q

Describe potential hazards of genetically manipulating pharmaceutical proteins

A
  • There are concerns for the welfare of the GM sheep and goats
    However, these animals are valuable and likely to be well looked after
85
Q

Describe potential benefits of genetically manipulating silk

A
  • Silk is one of the strongest materials known
  • Spiders are impossible to farm but genes for spider silk have been inserted into goats
  • These GM goats produce spider silk protein in their milk
  • Silk can be used for cables, sutures, artificial ligaments and bullet proof vests
86
Q

Describe potential hazards of genetically manipulating silk

A
  • Concerns were raised about the welfare of the
    GM goats
    However, these animals are valuable and likely to be well looked after and not be eaten
87
Q

Briefly outline genetic engineering

A
  • also known as recombinant DNA technology as it involves combining DMA from different organisms
  • also called genetic modification
  • genes are isolated from one organism and inserted into another organism using suitable vectors
88
Q

What are the main stages of genetic engineering

A

1) required gene is obtained
2) a copy of they gene is placed inside a vector
3) the vector carries the even into a recipient cell
4) The recipient expresses the novel gene

89
Q

Name different methods of obtaining the required gene in genetic engineering

A

Using mRNA:
- mRNA obtained from cells where gene is being expressed
- reverse transcriptase enzyme can catalyse formation of single strand of complimentary DNA (cDNA) using the mRNA as a template
- addition of primers can DNA polymerase can make this cNA into. double-stranded length of DNA, whose base sequence codes for the original protein

Machine:
- if scientists know the nucleotide sequence, the gene can be synthesised using an automated polynuceotide synthesiser

PCR:
- if scientists know the sequence, they can design PR primers to amplify the gene from teh genomic DNA

Probe:
- DNA probs can be used to locate a gene within the genome and the gene can be cut out restriction enzyme

90
Q

Name different methods of placing the gene into a vector in genetic engineering

A
  • plasmids can be obtained from organisms (e.g. bacteria) and mixed with restriction enzymes that will cut the plasmid at specific recognition sites
  • the cut plasmid has exposed unpaired nucleotide bases- sticky ends
  • if free nucleotide bases (complimentary to the sticky ends of the plasmid) are added to the ends of the gene to be inserted, then then gene and cut plasmid should anneal- catalysed by DNA ligase
  • a gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell
91
Q

Name different methods of getting the vector into the recipient cell, and say why these are needed (genetic engineering)

A
  • heat shock treatment
  • electroporation
  • electrofusion
  • transfection
  • T1 plasmids

Needed as DNA doesn’t easily cross the recipient cells plasma membrane

92
Q

Describe heat shock treatment as a method of getting the vector into the recipient cell

A
  • if bacteria subject to alternating periods of cold (OoC) and heat (42oC) in the presence of calcium chloride, their walls and memranes will become more porous and allow in the recombinant vector
  • happens as he positive claim ions surround the negatively charged parts of both the DNA molecules and the phospholipids in the cell membrane- reducing repulsion between the foreign DNA and the host cell membranes
93
Q

Describe electroporation as a method of getting the vector into the recipient cell

A

A high voltage pulse is applied to the cell to disrupt the membrane

94
Q

Describe electrofusion as a method of getting the vector into the recipient cell

A

Electrical fields help to introduce DNA into cells

95
Q

Describe transfection as a method of getting the vector into the recipient cell

A
  • DNA can be packaged inti a bacteriophage- can then transfect the host cell
96
Q

Describe T1 plasmids as a method of getting the vector into the recipient cell

A
  • T1 (recombinant) plasmids are inserted into the bacterium agrobacterium tumefciens- infects some plants and naturally inserts its genome into the host cell genomes
97
Q

Describe a direct method of introducing the gene into the recipient in genetic engineering

A
  • if plants are not susceptible to A. tumefaciens, direct methods an be used
  • small pieces of gold or tungsten are coated with the DNA and shot into the plant cells- called a gene gun
98
Q

Outline the role of reverse transcriptase in genetic engineering

A
  • retroviruses, such as HIV, contain RNA that they inject into the host genome, have reverse transcriptase enzyme that catalyses the product on cDNA using ether RNA as a template- reverse trasncription
99
Q

Outline the role of restriction enzymes in genetic engineering

A
  • bacteria and archaea have restriction endonuclease- protect them from attack by phage viruses- cut up teh foreign viral DNA- process called restriction- prevents viruses from making copies of themselves
  • the prokaryotic DNA is protected from the acton of these endonuclease by being methylated at the recognition sites
  • named according to bacterium from which obtained (e.g. fist one EcoR1 obtained from E.coli- restriction endonuclease 1)
  • some need magnesium ions as cofactors
  • act as molecular scissors- recognise specific sequences within a length of DNA and cleave the molecules there
100
Q

Describe different ends left by restriction enzymes

A
  • some make staggered cut leave sticks ends
  • some leave blunt ends
  • always recognise a palindromic sequence- reading the 2 strands of DNA in the same orientation (e.g. 5’ to 3’)- sequence of bases is the same
101
Q

what do some restriction enzymes need to function

A

magnesium ions as cofactors

102
Q

Outline the role of ligase enzymes in genetic engineering

A
  • DNA ligase is used in molecular biology to join DNA fragments
  • catalyses the condensation reactions that join the sugar groups and phosphate groups of the DNA backbone
  • these enzymes catalyse such reactions during DNA replication in cells, used in the PCR
103
Q

Describe how insulin is made from genetically modified bacteria

A

1) Beta cells from islets of langerhans in human Pancras are obtained
2) Add reverse transcriptase enzyme- makes a single strands of cDNA
3) treat with DNA polymerase- makes a double strand (the gene)
4) Addition of the unpaired nucleotides at the ends of the DNA produces sticky ends
5) Use DNA ligase to insert the insulin gene into the plasmids extracted from the E.coli bacteria- called recombinant plasmids as contain inserted DNA
6) E.coli bacteria mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions will take up the plasmids

104
Q

What are two things that can go wrong in producing insulin from genetically modified bacteria

A
  • plasmid may not take up ten gene
  • bacteria may not take up the recombinant plasmid
105
Q

Describe how insulin-producing genetically modified bacteria is tested for whether it worked

A
  • plasmid contains an ampicillin-resistance gene, and a tetracycline-resistance gene- the TR gene is at the location in which the human insulin gene is inserted
  • first, the plasmid is placed in ampicillin agar- if they all survive, you know that the bacteria has taken up the whole plasmid
  • next, they are placed on tetracycline agar- if any survive, the plasmid itself HASN’T taken up the human insulin gene as the resistance gene hasn’t been interfered with
  • thus, a successful bacteria should survive on the ampicillin agar (as means plasmid has taken) but should not survive on the tetracycline agar (as means gene has taken)
106
Q

Describe safety in producing insulin from genetically modified bacteria

A
  • transformed (transgenic) bacteria have resistance to some antibiotics- don’t want them to escape from labs into the wild
  • also have a gene knocked out- man they can’t synthesise a particular nutrient
  • can survive in laboratory where they are given that nutrient in their growth medium, but will not survive outside of the laboratory
107
Q

What’s the principle of gene therapy

A
  • to insert a functional allele of a particular gene into ells that contain only mutated and non-functioning alleles of that gene
  • if the inserted allele is expressed, the individual will produce a functioning allele and no longer have the symptoms associated with the genetic disorder
108
Q

Describe development in gene therapy

A
  • knowledge gained from the human genome project has led to further possibilities e.g. using interference RNA to silence genes by blocking translation
  • has been used to treat cytomegalovirus infections in AIDS patients by blocking replication of the cytomegalovirus
109
Q

name 2 types of gene therapy

A
  • germ line gene therapy
  • Somatic cell gene therapy
110
Q

Define somatic cell gene therapy

A
  • gene therapy by inserting functional alleles into body cells - affects only certain cell types- alterations made to the patients genome in this cells are not passed to the patients offspring
111
Q

Define germ line gene therapy

A

Gene therapy by inserting functional alleles into gametes or zygotes- all the cells of that individual will be altered, offspring may inherit the foreign alleles- potential to change genetic makeup of many people

112
Q

Describe applications/brief methodology of somatic cell gene therapy

A
  • some metabolic disorders e.g. cystic fibrosis occur when an individual inherence 2 recessive alleles for particular gene- different cells where this gene should normaly be pressed lack the portein product of that gene- if the functioning alleles for these gene can be put into specific cells so taht these cells then make the protein, the cells will function normally
113
Q

Name different methods of delivering the functioning alleles yo the patients body cells in somatic cell gene therapy

A
  • liposomes
  • viruses
  • artificial chromosomes
114
Q

Descrive treatment of cystic fibrosis using somatic cell gene therapy

A
  • lack a functioning CFTR gene
  • the allies can be packaged into liposomes
  • these are placed in aerosol inhaler and sprayed into the noses of patients, some will pass through the plasma membrane of cells lining the respiratory tract
  • if they also pass through the nuclear envelope and insert int the host genome, the host cell will express the CFTR protein- a transmembrane chloride ion channels
  • epithelial cells lining respiratory tracy are replaced every 10-14 days, so has to be repeated at regular short intervals
115
Q

Describe the use of liposomes in somatic cell gene therapy

A
  • small spheres of laid bilayer- have length of DNA (allele) packaged in it- can cross plasma membrane and nuclear envelope
116
Q

Describe the use of viruses in somatic cell gene therapy

A
  • used as vectors
  • virus that usually infects humans is genetically modified so that it encases he functioning allele to be inserted into the patient, whilst also being made unable to cause a disease, so it can enter the recipient cells- take the allele with it
117
Q

What are issues with using viruses in somatic cell gene therapy

A
  • in 1999, patent taking part in trial for this technique died
  • in 2002, trials interrupted as several patients developed leukaemia
  • 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
  • virus may insert the allele into the patient genome in a location. that disrupts a gene involved in regulating cell division-increasing risk of cancer
  • it may insert the allele into a patients genome in a location that disrupts the regulation of the expression of other genes
118
Q

Describe the use of artificial chromosomes in somatic cell gene therapy

A
  • research being carried out into possibility of insuring genes into an artificial chromosome that would co-exist with the other 46 chromosomes in the target cells
119
Q

What are issues with germ-line gene therapy

A
  • has potential to change genetic makeup of many people- descendants of original patient- none of whom would have given consent
  • how gene is inserted- may find their way into a location that could disrupt the expression or regulation of other genes or increase risk of cancer
  • means strict guidelines drawn up by regulatory bodies and ethics committees consider germ-linen gene therapy for humans to be ethically impermissible

Module 6- Genetics and Ecosystems (6 decks)

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