6.3 Flashcards
(165 cards)
1
Q
what happened in DNA research in 1969 and then in 1972
A
a gene was isolated from a bacterial chromsome, in 1972 a scientist sequenced a gene that codes for the protein coat of a virus. Both scientits worked from mRNA transcribed from the gene and not the raw DNA. RNA is unstable and this whole process was extrwemly slow and only worked for short genes
2
Q
what happened in DNA research in 1975
A
the biochemist Sanger develpoed a method that ultimately allowed scientists to sequence whole genomes
3
Q
what was Sanger’s approach
A
to use a single strand of DNA as a template for 4 experiments in separate dishes, each dish contained a solution with the 4 bases (A,T,G,C), plus an enzyme (DNA polymerase)
4
Q
what was added to each dish in Sanger’s DNA sequencing approach
A
a modified version of one of the DNA bases was added (ddNTP), the base was modified in a way that, once incorporated into the synthesised complementary strand of DNA, no more bases could be added, each modified was was also labelled with a radioactive isotope.
5
Q
whaqt happens in sanger’s DNA sequencing approach as the reaction progressed
A
thousands of DNA fragments of varying lengths were generated, the DNA fragments were passed trhough a gel by electrophoresis. Smaller fragments travalled further, so the fragments became shorted by length
6
Q
how were the nucleotide base at the end of each fragment read in Sangers DNA sequencing
A
by its radioactive label. If the first one-base fragment has tymine at the end, then the first base in the sequence is T. If the two-base fragments have cytosine at the end, then the sequece is TC. If the three-base fragment ends with guanine, then the base sequence is TCG.
7
Q
why did Sanger’s DNA sequencing work and what was it first used for
A
his method is efficient and safe. He first used it to sequence the genome of a phage virus (virus that infects bacteria cells) called Phi-X174, the first DNA based organsim to have its genome sequenced. He has to count off the bases one by one, from the bands in the gel (time consuming and costly process)
8
Q
what did SAnger do in 1981, 1984 and what happened in 1995
A
sanger published his sequence of the human mitochondrial genome, consisting of 37 genes and 16 569 base pairs. In 1984, scientists sequenced 170 kilobase pair-long genomes of the Epstein-Barr virus. In 1995, the genome of the bacterium Haemophilus influenzae was sequenced using this approach.
9
Q
In Sangers DNA sequencing how was the gene isolated (cloning DNA) and then what happened with the isolated gene
A
using restricted enzymes from a bacterium, the DNA was then inserted into a bacterial plasmid (the vector) and then into an Eschuerichia Coli bacterium host that, when cultured, divided many times, enabling the plasmid with the DNA insert to be copied many times.
10
Q
what did each new bacterium contain when cloning DNA using Sanger’s DNA sequencing and how were these lengths of DNA isolated
A
a copy of the candidate gene, these lengths of DNA were isolated using plasmid preparation techniques and then they were sequenced
11
Q
what happened with DNA sequencing in 1986
A
the first DNA sequencing machine was developed, based on Sanger’s method. Fluorescent dyes instead of radioactivity were used to label the terminal bases, these dyes glowed when scanned with a laser beam and the light signature was identified by computer. This method needed technitions to read autogradiograms.
12
Q
what is high throughput sequencing and give an example
A
an approach to develop fast, cheap methods to sequence genomes. an example is pyrosequencing
13
Q
what is pyrosequecnig and what does it involve
A
developed in 1996 and uses sequencing by synthesis, not by chain termination as in the Sanger method. It involves synthesising a single strand of DNA, complementary to the strand being sequenced, one base at a time, whilst detecting, by light admission, which base was added to each step.
14
Q
what are steps 1-2 of pyrosequencing
A
- 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). These are the template DNAs and they are immoblisied.
15
Q
what is step 3 of pyrosequencing
A
- a sequencing primer is added and the DNA is incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin, only 1 of the 4 possible activated nucleotides, ATP, TTP, CTP and GTP is added at any one time and any light generated is detected.
16
Q
what is step 4 of pyrosequencing
A
1 activated nucleotide (a nucleotide with 2 extra phosphoryl groups), like TTP (thymine triphosphate), is incorporated into a complementary strand of DNA using the strand to be sequenced as aa template. As this happens the 2 extra phosphoryls are released as pyrophosphate (PPi). In presence of APS, the enzyme ATP sulfurylase converts phosphate to ATP. In presence of this ATP, enzyme luciferase converts luciferin to oxyluciferin. This conversion generates visible light that can be detected by a camera. The amount of light generated is proportional to ATP availability and so indicates how many of the same type of activated nucleotide were incorporated adjacently into complementary DNA strands.
17
Q
what happens to unincorporated activated nucleotides in pyrosequencing
A
they are degraded by apyrase and the reaction starts again with another nucleotide.
18
Q
how long does pyrosequencing take
A
10h run generate 400 million bases of sequencing information which are assembled into longer sequences by software.
19
Q
what is bioinformatics
A
a branch of biology that has grown out of DNA sequencing research, to store the huge amounts of data generated. Prior to computers, it was impossible to store all this information but software packages are specially designed for this purpose.
20
Q
what is the human genome project
A
scientists predicted human genome would contain 100 000 genes. In 1990, the human genome project launched and the genome was sequenced by 2003. Scientists were surprised to learn human genome only contained 24 000 genes (not too many more than a mouse)
21
Q
what dies whole genome sequencing determine
A
the complete DNA sequence of an organsism’s genome, in the case of eukaryote cells, that is the genetic material of the chromosomes, mitochondria and, if plants or algae, chloroplasts.
22
Q
where are sequenced genomes stored
A
gene banks
23
Q
What happened when human genome was compared to other species
A
it was clear that few human genes are unique to us, most of our genes were present in other organisms, we share 99% of our genes with chimpanzee, this verifies the evolutionary process
24
Q
what is an examlpe of how comparism of genome has provided useful
A
pigs and humans have similar genes for insulin, which is why, prior to genetically-modifying bacteria to make insulin, pig insulin was used to treat patients with diabetes
25
what sometimes happpens as evolution progresses
some genes are co-opted to perform new tasks
26
what has a tiny change to a gene in humans allowed
tiny changes in human FOXP2, which is found in other mammals including mice and chimpanzees, means that humans can speak
27
How are differences between organisms explained at a gene level
there not different because they have completely different genes, but as some of their shared genes have been altered and no work in slightly different ways
28
what has altered the expression of genomes
some changes to the regulatory regions of DNA that don't directly code for proteins, regulatory coding and genes interact in a way that, without increasing the number of genes, the number of proteins may be increased.
29
what has comparing genomes of thought to be closely related organisms allowed
helped confirm their evolutionary relationship or led to new info about their relationship, some cases led to organisms being reclassified.
30
how can animals evolutionary history be verified
DNA from bones and teeth of some extinct animals can be amplified and sequenced
31
what was found when extinct cave bears genome was sequenced using high throughput techniques
obtained a comparison to dogs, dogs and cave bears shared 92% of their genome
32
Are all humans genetically similar
yes, unless in rare cases where a gene has been lost by deletion of part of a chromosome, we all have the same genes, but different alleles
33
how much of out DNA is not shared with others and what does this mean
only 0.1%, although this sounds small, given that our genome contains 2 billion DNA base pairs, means there are 3 million places on DNA length where our sequences can differ due to random mutation
34
what is the name of the places on the DNA where these substitutions occur
single nucleotide polymorphisms, or SNPs
35
what is the effect of single nucleotide polymorphisms
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
36
What dies methylation of certain chemical groups in DNA play a major role in
regulating gene expression in eukaryote cells
37
what does mapping methylation of whole human genomes help researchers to understand
the development of certain diseases, like certain cancers and why they may not develop in genetically similar individuals. The study of this is called epigenetics
38
how easy is it to determine sequencde of amino acids in a protein
time consuming and laborius
39
how do researchers work out the primary structure of a protein
if they have the genome and know which genes code for a specific protein, by using knowledge of which base triplets code for which amino acids, they can determine primary structure, researchers need to know which part of the gene codes for exons and which for introns
40
what is synthetic biology
an interdisciplinary science concerned with designing and building useful biological devices and systems, it includes biotechnology, evolutionary biology, molecular biology, systems biology and biophysics. Its goal is to build engineered biological systems that store and process information, provide food, maintain human health and enhance the environment
41
what does the sequences of DNA found by analysis genome provide
potential building blocks for synthetic biologists to build devices
42
what is information storage as an example for synthetic biology application
scientists can encode a vast amount of digital info onto a single strand of synthetic DNA
43
what is production of medicine as an application for synthetic biology
E.Coli and yeast have both been genetically engineered to produce the precursor of a good antimalarial drug, previously only available by extracting from certain parts of a plant at particular times in its life cycle
44
what is novel protiens as an application for synthetic biology
designed proteins have been produced, like one that is similar to haemoglobin and binds to oxygen, but not to carbon monoxide
45
what is biosensors as an application for synthetic biology
modified bioluminescent bacteria placed on microchip coating glows if the air is polluted with petroleum pollutants
46
what is nanotechnology as an application for synthetic biology
material can be produced for nanotechnology
47
what is bioethics
synthetic biology raises issues of ethics and security, extensive regulations are in place after 30 years of using genetically-modified organisms, many advisory panels and many papers are written on how to manage risks. Synthetic biology isn't about synthesising life from scratch, but the potential for new systems with rewards and associated risks to be managed.
48
what was jeffreys doing in 1978
locating tandem repeat sequences of DNA
49
what are tandem repeats
repeptitative segemnts of DNA that do not code for proteins. They may be between 10-100 base pairs long and all feature the same core sequence, GGGCAGGAXG, where X can be any of the 4 nucleotide bases.
50
Where do tandem repeats occur
at more than 1000 locations in the genome and, in each of these places, they may be repeated a random number of times
51
what are variable number tandem repeats (VNTRs)
highly variable tandem repeats
52
what did Jeffreys obtain in 1978
some DNA from his lab technician and her parents and analysed it.
53
Why was Jefferys suprised with the DNA he obtained in 1978
the number of tandem repeats showed a family resemblance, but the DNA profile for each family member was unique. He realsied that a person's DNA profile could confirm or refute maternity or paternity
54
What is steps 1-2 of DNA profiling
1. DNA is obtained from the individual by mouth swab, from saliva on toothbrush, from blood or hair or from bones in case of ancient remains 2. the DNA is digested with restriction enzymes, these enzymes cut the DNA at specific recognition sites, they will cut it into fragments, which vary in size from individual to individual
55
What is steps 3-4 of DNA profiling
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
56
What is steps 5-6 of DNA profiling
5. the DNA to which individual is being compared is treated with same restriction enzymes and also subject to electrophoresis 6. the banding patterns of the DNA samples can then be compared
57
What did the first method of DNA profiling involve and why was it adapted
involved restriction fragment length polymorphism analysis which is laborious and no longer used
58
what is now used when profiling DNA
short tandem repeat (STR) sequences of DNA are used. These are highly variable short repeating lengths of DNA, the exact number of STRs varies person to person
59
how are short tandem repeat (STR) sequences separated
by electrophoresis. Each STR is polymorphic but the number of alleles in the gene pool for each one is small. 13 STRs are analysed simultaneously, so although each STR is present in 5-20% of individuals, the chance of 2 people sharing STR sequences at all the loci is 1x10*18.
60
This short tandem repeat sensistive or not and what does this mean
it is very sensitive and even a trace of DNA left when someone touches an object can produce a result, samples must be treated carefully to avoid contamination
61
how long can DNA be stored for and when is it useful to do so
for many years, in unsolved crimes can be used as evidence years later
62
How has DNA profiling transformed forensic science
brought about convictions and established victims who were previously convicted of crimes
63
what are some examples of how DNA profiling has been used in forensic science
identitfy Nazi war criminals is south america, identify victims body parts after air crashes, terror attacks or other disasters, match profiles from descendants of those lost in WW1
64
What is a childs genetic infomation made up of and what does this mean
half mums and halfs dads genetic infomation, so half of short tandem repeat fragments come from mother and half from father. This means comparing DNA profiles of mum, dad, child can determine materinity and/or paternity
65
what can protein electrophoresis detect
the type of haemoglobin present and aid diagnosis of sickle cell anaemia, a varying number of repeat sequences for a condition like huntingtons can also be detected by electrophoresis
66
what are some limitations of analsying DNA from crime scenes
if a few cells are left behind the criminals DNA could be obtained but an innocent person could have touched the same surface and be wrongly convicted
67
What did Mullis develop in 1983
the polymerase chain reaction (PCR) to amplify the amount of DNA, enabling it to be analysed
68
what is PCR
artificial DNA replication
69
what does PCR rely on
facts that DNA is made up of 2 antiparallel backbone strands, each starnd has a 5' and 3' end, DNA grows only from 3' end, base pairs pair up according to complementary base pairing rules
70
how does PCR differ from DNA replication
only short sequences, 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 needed to separate DNA strands, bind primers to the strands and for the DNA strands to be replicated
71
what reaction is a PCR
cyclic reaction
72
why was the first PCR method time consuming
the DNA was heated to denature it and then cooled to around 35degrees to anneal the primers and allow DNA polymerase to work
73
how did PCR method develop
DNA polymerase was obtained from a thermophilic bacterium, this enzyme is called Taq polymerase and is stable at high temperatures
74
what are steps 1-2 of PCR method
1. sample of DNA is mixed with DNA nucleotides, primers, magnesium ions and enzyme Taq DNA polymerase 2. the mixture is heated around 95 degressC to break hydrogen bonds between complementary nucleotide base pairs and thus denatures the double-stranded DNA into 2 single strands of DNA
75
what are steps 3-4 of PCR method
3. mixture is cooled to around 68degreesC, so that the primers can anneal (bind by hudrogen bonding) to one end of each singke strand of DNA, this 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 doubled stranded DNA, Taq polymerase obtained from a bacterium that lives in high temps, 72degreesC is optimum for this enzyme
76
what are steps 5-6 of PCR method
5. the temp is raided to 72degreesC, which keeps the DNA as single strands 6. Taq DNA polymerase catalyses the addition of DNA nucleotides to single-standed DNA molecules, starting at end with the primer and proceeding in the 5' to 3' direction
77
what are steps 7-8 of PCR method
7. when Taq 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
78
in PCR how does the amount of DNA increase
exponentially, 1->2->4->8->16->32->64->128...
79
how is tissue typing an application of PCR
donor and recipient tissues can be typed prior to transplantation to reduce the risk of rejection of the transplant
80
how is detection of oncogenes an application of PCR
if the type of mutation involved in a specific patients cancer is found, then the medication may be better tailored to that paitent
81
how is detecting mutations an application of PCR
a sample of DNA is analysed for the presence of mutations that cause genetic diseases, parents can be tested to see if they carry a recessive allele for a certain gene, fetal cells obtained from mother blood stream for prenatal genetic screening or during IVR treatment
82
how is identifying viral infections an application of PCR
sensistive PCR tests can detect small quantities of viral genome amongst the host cells' DNA, this can be used to verify HIV or hepatitis C infections, for example
83
how is ,onitoring spread of infectioius diseases an application of PCR
spread of pathogens through a population can be monitored and emergence of new more virulent sub-types can be detected
84
how is forensic science an application of PCR
small quantites of DNA can be amplified for DNA profiling to identify criminals or ascertain parentage
85
how is research an application of PCR
amplyfiing DNA from extinct ancient sourcees like neanderthal bones for analysis and sequencing, in extant organisms, tissues or cells can be analysed to find out which genes are switched on and off
86
what is electrophoresis used to do
separate different sized fragments of DNA, it can separate fragments of DNA that differ by only 1 base pair and is widely used in gene technology to separate DNA fragments for identification and anlysis
87
what does the electrophoresis technique use
an agarose gel plate covered by a buffer solution
88
how does electrophoresis work
electrodes are placed in each end of the tank so that when its connected to a power supply, an electric current can pass through the gel, DNA has an overall negative charge, due to its many phospahte groups, and the fragments migrate towards the anode (positive electrode), fragments of DNA all have a similar surface charge regardless of their size
89
what is steps 1-2 of electrophoresis
1. the DNA samples are first digested with restriction enzymes to cut them, at specific recognition sites, into fragments, carried out at 35-40degrees and may take an hour 2. whilst restriction enzymes cutting DNA, the tank is set up, agarose gel made up and poured into central region of tank whilst combs are in place at one end, once the gel is set, buffer solution is added so gel is covered and end sections of the tank contain buffer solution, now comb is carefully removed leaving wells at 1 end of the gel
90
what is steps 3- 4 of electrophoresis
3. a loading dye is added to the tubes containing the digested DNA 4. digested DNA plus loading dye is added to wells in an electrophoresis gel, to do this a pipette is used and this is held in the buffer solution just above one of the wells, loading dye is dense and carries DNA down into the well, the pipette should not be placed right into the well, otherwise you may pierce the bottom of the well
91
what is steps 5-6 of electrophoresis
5. once all the wells have been loaded with different DNA samples, the electrodes are put into place and connected to an 18V battery, this is then left to run for 6-8hours or a high voltage power pack can be used and gel will run for only 2h 6. DNA fragments move through gel at different speeds, smaller fragments travel faster so travel further in a fixed period
92
what is step 7 of electrophoresis
7. at 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
92
what is step 7 of electrophoresis
7. at 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
92
what is step 7 of electrophoresis
7. at 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
93
how is separating proteins different from DNA with electrophoresis
its the same but often carried out in the presence of a charged detergent like sodium dodecyl sulfate (SDS) which equalised the surface charge on the molecules and allows proteins to separate as they move through the gel according to molecular mass
94
why conditions can be detected by analysing haemoglobin proteins using electrophoresis
sickle cell anemia, where paitent has haemoglobin S and not the normal haemoglobin A, and, aplastic anaemia, thalassaemia and leukaemia, where patients have higher than normal amounts of fetal haemoglobin and lower amount of haemoglobin A
95
what is a DNA probe
a short (50-80 nucleotides) single-stranded length of DNA that is complementary to a section of the DNA being investigated
96
how may a DNA probe by labelled (2 ways)
by a radioactive marker, usually with *32P in 1 of the phosphate groups in the probe strand, once the probe has annealed (bound), by complementary base pairing, to the piece of DNA, it can be revealed by exposure to photographic film to by a fluorescent marker that emits a colour on exposure to UV light, may also be used in automated DNA sequencing
97
what aare 3 examples where DNA probes are useful in locating specfic DNA sequences
to locate a specific gene needed for use in genetic engineering, to identify same gene in many different genomes from different species when conducting genome comparison studies, to identify the presence or absence of a specific allele for a certain genetic disease or that gives susceptibility to a certain condition
98
What are DNA microarrays and what is thier purpose
scientists can place a number of different probes on a fixed surface (microarrays), applying DNA under investigation to the surface can reveal the presence of mutated alleles that match the fixed probes as the sample DNA will anneal to any complementary fixed probes
99
What must occur before a microarray can be made
sample DNA must be broken into smaller fragments and amplified using polymerase chain reaction (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.
100
how are reference and test DNA samples analysed in microarrays
samples are labelled with fluorescent markers, where a test subject and a reference marker both bind to a particular probe, the scan reveals fluorescence of both colours, indicating the presence of the particular sequence in the test DNA
101
what is genetic engineering also known as and why
recombinant DNA technology because it involves combining DNA from different organisms, it is also called genetic modification as genes are isolated from one orgnaism and inserted into another using a suitable vector
102
what are the 4 necessary stages of genetic engineering
1. required gene is obtained 2. copy of gene placed inside vector 3. vector carries the gene into a recipient cell 4. recipient expresses the novel gene
103
how is the gene obtained in genetic engineering
mRNA can be obtained from cells where the gene is being expressed, enzyme, reverse transcriptase, catalyses the formation of a single strand pf complementary DNA (cDNA) using mRNA as a template. Addition of primers and DNA polymerase makes the cDNA into a double-stranded length of DNA, whose base sequence can code for the original protein
104
if scientists know the nucleotide sequence of the gene, how is the required gene obtained for genetic engineering
gene is synthesised using an automated polynucleotide synthesiser
105
if scientists know the sequence of the gene, how is the required gene obtained for genetic engineering
they can design polymerase chain reaction (PCR) primers to amplify the gene from the genomic DNA
106
how can a DNA probe be used to obtain required gene for genetic engineering
DNA probe can be used to locate a gene from within the genome and the gene can then be cut out using restriction enzymes
107
How is a gene plaved into a vector in genetic engineering
plasmids can be obtained from bacteria and mixed with restriction enzymes that cut the plasmid at specifc recognition sites, cut plasmid exposes sticky ends (unpaired nucleotide bases). If free nucleotide bases, complemntray to sticky ends, are added to end of the gene to be inserted then the gene and cut plasmid should bind (anneal), DNA ligase catalyses the binding. Gene may be sealed into an attenuated virus that could carry it in a host cell
108
how does getting the vector into the recipient cell work in genetic engineering
DNA doesn't easily cross recipients cell plasma membrane, but various methods used to help this
109
what is heat shock method as a way to get the vector into recipient cell in genetic engineering
iif bacteria subjected to alternating periods of cold (0degreeC) and hot (42degreesC) in presence of calcium chloride, their walls and membranes become more porous and allow the recombinant vector. This is as the positive calcium ions surround the negativeky charged parts of both DNA molecules and phospholipids in the cell membrane, so reducing repulsion between the foreign DNA and hosts cell membranes
110
what is electroporation as a way to get the vector into recipient cell in genetic engineering
a high voltage pulse is applied to the cell to disrupt the membrane
111
what is electrofusion as a way to get the vector into recipient cell in genetic engineering
electrical fields help to introduce DNA into cells
112
what is transfection as a way to get the vector into recipient cell in genetic engineering
DNA can be packaged into a bacteriophagewhich can then tranfect the host cell
113
what is T1 (recombinant) plasmids as a way to get the vector into recipient cell in genetic engineering
T1 (recombinant) plasmids are inserted into a bacterium which infects some plants and naturally inserts its genome into the host cell genomes
114
what happens if plants are not suceptibale to the bacteria with the T1 (recombinant) plasmids
direct methods are used, small pieces of gold or tungsten are coated with DNA and shot into the plant cells, called a 'gene gun'
115
How are reverse transcritase enzymes useful for genetic engennering
retroviruses like HIV, which contain RNA that they inject into the hosts genome have reverse transcriptase enzymes that catalyse prouction of cDNA using their RNA as a template, this is the reverse of transcription
116
How are restriction enzymes useful for genetic engennering
Bacteria and Archaea have restriction enzymes, called restriction endonuclease, to protect the form attack by phage viruses, the enzymes cut up foreign viral DNA by a process called restriction, preventing the virus from making copies of themselves, the prokaryotic DNA is protected from the action of these endonuclease by being methylated at the recognition sites
117
what are restriciton enzymes useful to
molecular biology and biotechnology as molecular scissors as they recognise specific sequences within a length of DNA and cleave the molecule there, some make a staggereed cut leaving sticky ends and others make a cut producing blank ends (no free nucleotide bases)
118
How are ligase enzymes useful for genetic engennering
used in molecular biology to join DNA fragments, it catalyses comsdensation reacxtios that join the sugar groups and phospahte groups of the DNA backbone, these enzymes catalyse these reactions during DNA replication in cells or used in PCR
119
what are the first 2 steps to obtain mRNA from beta cells of islets of langerhans in human pancreas where insulin is made to help form insulin from GM bacteria
1. adding reverse transcriptase enzyme makes a single strand of cDNA and treatment with DNA polymerase make a double strand - the gene 2. addition of free unpaired nucleotides at end of DNA produces sticky ends
120
what are the last 2 steps to obtain mRNA from beta cells of islets of langerhans in human pancreas where insulin is made to help form insulin from GM bacteria
3. now woth help of ligase enzyme, insulin gene can be inserted into plasmids extractced from E.coli bacteria, now called recombinat plasmids, as they contain inserted DNA 4. E.coli bacteria are mixed with recombinant plasmids and subjected to heat shock in presence of calcium chloride ions, so that they will take up the plasmids. Genetically modified bacteria are then cultured in large numbers to produce insulin, recombinant plasmids grow on namplicillin agar
121
What safety precautions must be made when syntheissing insulin from GM bacteria
as transgenic bacteria have resistance to some antibiotics we don't want them to escape form labs, so they have a gene knocked out so they can't synthesise a particular nutrient, they survive in lab when they are given this nutrient in their growth medium but will die outside of the lab.
122
what has happened to crops grown around the world as a result of human inervention and selective breeding
they have got vastly different genomes from their wild relatives
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WHat has agriculture caused
a change in the face of the landscape and produced domesticated breeds of animals and plants
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what is a limitation of selective breeding
it can be a hit and miss technnique and produce unexpected results
125
What did the technique of recombinant DNA technology allow
scientists to splice new genes into plant genomes giving more exact way of transforming crop plants, compared with inducing mutations or crossing different strains, now specific genes with desirable traits can be excised from 1 organsim and inserted into another, using a vector or 'gene gun'
126
What are opinions of gene modification
some are concerned about potential hazards and risks associated with it, but, potential benefits have to be recognised and weighted against potential hazards. Anti-GMers want technology abolished but this reduces potential benefits
127
What are benefits of GM microorganisms
can make human insulin and human growth hormones to treat pituitary dwarfism
128
What are hazards of GM microorganisms
could escape into wild and transfer antibiotic resistant to other bacteria (but have been edited so can't survive outside the lab)
129
What are benefits of GM plants
tobacco plants modified to produce toxin normally prodiced by bacteria, used by farmers as a pesticid. Bacterial gene was inserted into some plants, GM plants naturally produce the toxin, eliminating the need for spray
130
What are hazards of GM plants
toxic to monarch butterflies, however, they do not need nutriets from tobacco plants so hasn't effected their species
131
What are benefits of GM soya beans
resistant to a herbicide, so weeds competing with soya beans can be killed with herbicide
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What are hazards of GM soya beans
gene herbicide resistance to pass into weeds
133
What are benefits of GM golden rice
many children in less developed countries go blind due to vitamin A, golden rice GM to increase amount of vitamin A in it and since vitamin A deficiences is less of a problem
134
What are hazards of GM golden rice
people were concerned about the annual cost of these seeds, but company have developed seeds that can be replanted
135
What are benefits of GM plantains (type of banana)
plantains GM to produce more zinc, in areas where less meet is eaten, this extra zinc is essential for good insulin secretion
136
What are hazards of GM plantains (type of banana)
some people are concerned about eating GM food that inserted genes will be expressed in us, however this isn't true
137
What are benefits of GM crop plants resistant to pests
pesticides don't need to be used as crops have been produced that are resistant to pests, this is better for farmers and the environment
138
What are hazards of GM crop plants resistant to pests
concerns local farmers wouldn't want GM seeds and wouldn't be able to buy non GM seeds, but many farmers see benefits and non GM seeds still avaiable to buy
139
What are benefits of GM pathogens
Viruses GM to have no virulence, used to make vaccines or as vectors in gene therapy
140
What are hazards of GM pathogens
certain problems using GM viruses in gene therapy, can increase risk of cancer or interferes with gene regulation
141
What are benefits of GM mice
helps with medical research and develop therapies for cancer or to find functions of certain genes
142
What are hazards of GM mice
some people object to use of animals for research
143
What are benefits of GM pharmaceutical proteins
inserted into goat or sheep, and human protein they express in their milk is harvested, mammals used as protein too large for bacteriasl cells to synthesis
144
What are hazards of GM pharmaceutical proteins
concerns of welfare of GM sheeps and goats
145
What are benefits of GM silk
spiders cannot be farmed but protein for spider silk has been inserted in goats and this spider silk protein is produced in goats milk, silk can be used for cables, sutures, artifical ligamnets and bullet proof vests
146
What are hazards of GM silk
corcerns over welfare of GM goats
147
what is the basic principle of gene therapy
to insert a functional allele of a particular gene into cells that contain onlny mutated and non-functioning alleles of that gene, if the inserted allele is expressed, the individual will produce a functioning protein and no longer have symptoms associated with a genetic disorder
148
What has knowledge from human genome project led to within gene therapy
;more posibilities like interference RNA to silence genes by blocking translation, interference RNA has been used to create cytomegalovirus infections in AIDS patients by blocking its replication
149
when do some metabolic disorders like cystic fibrosis occur
when an individual inherits 2 faulty, recessive alleles for a particular gene
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what is the results when metabolic disorders like cystic fibrosis occur
differentiated cells where this gene should be expressed lack protein product of the gene, if functioning alleles for this gene can be put into specific cells so these cells can make the protein, they will functiuon normally
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what is somatic cell gene therapy
affects only certain types opf cells, alerations made to patients genome in those cells are not passed to pateints offspring
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what do pateints with cystic fibrosis lack
a functioning CFTR gene
153
what are made from liposomes
the alleles for gene therapy, lengths of DNA, can be packaged within small spheres of lipid bilayer to make liposomes
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how do lipososomes work in somatic cell gene therapy
liposomes placed into an aerosol inhaler and prayed into the nose of patients , some will pass through plasma membra`ne of cell lining into the respiratoy track. If they also pass the nuclear envelope and insert into the hosts genome, the host cell will express the CFTR protein (a transmembrane chloride ion channel)
155
why must somatic cell gene therapy with liposomes occur repeatedly at regular short intervals
epithelial cells lingings are replaced every 10-14 days
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how has viruses been used as vectors in somatic cell gene therpay
if a virus that usually infects humans in genetically modified so it encases the funcitonihng alleleto be inserted into the patient whilst not causing disease, it can enter the recipients cells, taking the allele with it
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what are 3 ways used to adminsiter somatic cell gene therapy
liposomes, viruses, artifical chromosomes
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what are 4 problemswith using viruses as gene delivery agents
viruses, even when not virulent, make promote immune resposne, patient may be immune to the virus making delivery impossible, virus may insert allele into patients genome in a location that disrupts a gene involved in regulating cell division, increasing risk of cancer, virus may insert allele into patients genome in location that disrupts regulariton of expression of other genes
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how has artifical chromosomes been used as vectors in somatic cell gene therpay
research being carried out looking at posibility of inserting genes into an artifical chromosome that would co-exist with the other 46 chromosmes in the target cells
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What does germ line gene therapy involve
altering genome of gametes or zygotes, not only will all cells of that individual be altered but their offspring will inherit foreign alleles
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what does germ line gene therapy have potential to do
to change genetic make up of many people, the descendants of the original patients who will not have consented
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why are there concerns about germ line gene therpay
how the gene is inserted-may find way into a location that could disrupt expression/regulation of other genes or increase risk of cancer
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why have strict guidlines been drawn up for germ line gene therapy
as for humans it is considered to be ethically impermissible
