Manipulating genomes 6.3 Flashcards
1
Q
Define DNA sequencing
A
DNA sequencing = a technique that allows genes to be isolated and read
2
Q
What did Fred Sanger do?
A
Fred Sanger developed a method that allowed scientists to sequence whole genomes through chain termination
3
Q
What was the proc and cons of fred sangers method?
A
DNA sequencing by chain termination
- safe
- efficient
- time consuming
- expensive
4
Q
What was the first step of chain termination?
A
DNA sequencing by chain termination
1.Single strand of DNA used.
4 dishes which contained a solution of A, T, C, G, DNA polymerase and a primer
5
Q
What was the second step of chain termination?
A
DNA sequencing by chain termination
2. modified DNA base labelled with radioactive isotope was added to the dishes. The base was modified so that once incorporated into the synthesis complementary strand of DNA, no more bases can be added. Chain stops
6
Q
What was the third step of chain termination?
A
DNA sequencing by chain termination
3.DNA fragments of varying lengths produced and passed through a gel by electrophoresis. Smaller fragments travel further so fragments get sorted by length
7
Q
What was the fourth step of chain termination?
A
DNA sequencing by chain termination
4. nucleotide base at the end of each fragment was read and you can then work out the base on the complimentary strand
8
Q
How do you separate DNA into single strands?
A
DNA is separated into single strands through constant heating
9
Q
Why is a primer needed in chain termination?
A
Primer is needed so that the DNA polymerase has something to bind to so that it can work
10
Q
How is DNA cloned?
A
Cloning DNA:
- gene isolated using restriction enzymes from bacterium
- DNA inserted into a bacterial plasmid (vector)
- plasmid inserted into E.coli bacterium host
- E.coli cultured + divides
- lengths of dna isolated using plasmid prePRTION
- DNA sequenced
11
Q
describe the first DNA sewurncing machine
A
First DNA sequencing machine:
- 1986
- used fluroscent dyes instead of radioactive isotopes
- scanned by laser beam
- light signature identified by a computer
- meant technicians didn’t need to waste time reading autoradiograms
12
Q
What is pyrosequencing?
A
Pyrosequencing reads the chain of DNA as you go along using one nucleotide at a time (instead of all at once in chain termination) I involves synthesising a ingle strand of DNA one base at a time and detecting light emission
13
Q
What are the basic stages of pyrosequencing?
A
Pyrosequencing:
- nubuliser cuts long DNA into fragments
- degraded to single strand DNA, act as template DNA which are immoiblised
- sequencing primer added and DNA incubated with 4 enzymes and 2 substrates
- one of the 4 activated nucleotide added at a time
- any light is detected by a camera
14
Q
Discuss light emissions from pyrosequencing
A
Pyrosequencing: amount of light detected is proportional to the amount of ATP available and therefore how many of the nucleotides incorporated successfully
- light = complimentary activated nucleotide has joined
- no light = no activated nucleotides have joined
15
Q
In the third stage of pyrosequencing what enzymes and substrates are added?
A
Pyrosequencing:
3. DNA incubated with enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase and the substartes; adenosine 5’ phosphosulfate (aps) and luciferin
16
Q
What does activated nucleotide mean?
A
Activated nucleotides have been phosphorylated, they have 2 phosphates joined on to them (e.g. TTP). When they join they release the 2 phophoryls pyrophosphate which gets converted to ATP
17
Q
What happens to unincorporated activated nucleotides?
A
Unincorporated activated nucleotides are degraded by apyrase
18
Q
Describe in detail the fourth step of pyrosequencing
A
Pyrosequencing:
- (a) Activated nucleotide is incorporated into a complimentary strand of DNA, using the strand to be sequenced as a template.
(b) The 2 phosphoryls are released as pyrophosphate
(c) In the presence of APS ATP sulfurylase converts the pyrophosphate to ATP
(d) In the presence of ATP luciferase converts luciferin to oxyluciferin which generates visible light which is detected by a camera
19
Q
What is the amount of light generated proportional to in pyrosequencing?
A
In pyrosequencing 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 complimentary DNA strand
20
Q
What are all of the activated nucleotides?
A
Activated nucleotides:
GTP, CTP. ATP, TTP
21
Q
What is the shorthand for single stranded DNA?
A
Single stranded DNA = ssDNA
22
Q
What is a pro of pyrosequencing?
A
Pyrosequencing is cheap and fast
23
Q
What is bioinformarics?
A
Bioinformatics is a bracnh of biology and its how the massive amounts of data generated from DNA sequencing is stored. It would have been impossible to store and analyse this data prior to the computers and microchips.
24
Q
What is electrophoresis?
A
Electrophoresis is the process used to spearate proteins or DNA fragaments of differwent sizes for identification and analysis
25
How is DNA digested before electrophoresis? Conditions?
DNA gets digested with restriction enzymes at special recognition sites into fragments. This is done at 35-40 degrees celcius and takes up to an hour
26
How is the tank for electrophoresis set up?
The tank has agarose gel poured into the centre and combs are placed at one end t create wells
27
What covers the agarose gel in electrophoresis?
Buffer solution is added to cover the agarose gel and end sections
28
What is added to the digested DNA in electrophoresis? Then whats done with it?
Loading dye is added to the digested DNA and this solution is hen pipetted into the buffer solution above the wells, then dense dye will carry the DNA into the wells
29
What voltage is the electrodes using and for how long? variation?
The electrodes are put into place and connected to a 18V battery for 6-8 hours. OR you use a higher voltage and leave for less that two hours
30
What is the overall charge of DNA and how does this affect electrophoresis?
The overall charge of DNA is negative due to the phosphate groups. So the DNA will move toward the anode (positive) and away from the cathode (negative)
31
What are the final two stages of electrophoresis? (how its actually separated)
DNA fragments move through gel at different speeds, smaller fragments go faster.
The buffer solution gets poured away and dye is added to the gel. The dye adheres to DNA and stains the fragments
32
Whats the difference between anodes and cathodes?
Anodes are positively charged
| Cathodes are negatively charged
33
Very briefly outline electrophoresis
Electrophoresis
1. digested with restriction enzymes
2. tank set up
3. loading dye added to DNA and pipette into buffer
4. Electrodes put into place 18V
5. DNA is negative and fragments different speeds
6. remove buffer solution
7. dye added to gel to adhere to fragments
34
In general what is the charge on proteins?
Proteins do not have the same charge all over but different areas of charge
35
Why is Sodium Dodecyl sulfate used for electrophoresis of proteins?
sodium dodecyl sulfate is used to equalise the surface charge of proteins as they do not have the same charge all over and denature them
36
What does sodium dodecyl sulfate allow the proteins to do?
Adding sodium dodecyl sulfate (equalizes surface charge) allows the proteins to separate by molecular mass as they move through the gel. Then they can move to the anode
37
How can proteins be separated without using sodium dodecyl sulfate?
Proteins can also be separated according to mass and then surface charge
38
How does electrophoresis of proteins and DNA differ?
DNA uses restriction enzymes to cut it up
| Proteins use sodium dodecyl sulfate to equalize and denature them so they can move and separate
39
What is electrophroesis of proteins used for?
Electrophoresis of proteins is used for analysing different types of haemoglobin proteins for diagnosing:
-sickle cell anaemia (have Hb S not Hb A)
- aplastic anaemia
- thalassaemia (more fetal Hb, less normal)
- leukaemia
40
What is a DNA probe?
A DNA probe is a short single stranded DNA length that is complimentary to a section of DNA being investigated
41
What are DNA useful fro/
DNA probes are useful for locating specific gene sequences
42
Why do DNA probes need to locate specific gene sequences?
Locating DNA sequences for :
- locating gene needed for genetic engineering
- identify the same gene in a variety of genomes from different species when doing genome comparison studies
- Identify presence or absence of a specific allele for a particular genetic disease/susceptibility for condition
43
What can the DNA probe be labelled with? How can these labels be seen?
The DNA probe labeling:
- Fluorescent marker that emits a colour on exposure to UV light
- Radioactive marker. P32 in one of the phosphate groups in the probe strand. Once annealed by complimentary base pairing to the DNA this can be exposed by photographic film
44
What is a micro array?
Micro array is a number of probes that are specific to sequences found in mutated alles that cause genetic disease on a fixed surface
45
What can applying DNA to a micro array reveal?
Applying DNA to a microarray can reveal the prescence of mutated alles as the DNA will anneal to any complimentary fixed probes
46
What is the process of using a microarray?
Using a microarray:
- Test DNA broken down into smaller fragments and amplified using the polymerase chain reaction
- Reference and test DNA labelled with fluorescent markers
- When a test subject and reference sample both bind to a probe of the same sequence the scan shows two colours indicating the prescence of a pasrtiular squece on the test DNA
47
What is reference DNA?
Refernece DNA has known mutated alleles that are comolimneary to the probe so this one will emit a flurorecence
48
What does 2 lights in the use of microarrays indicate and why?
Refernece DNA has known mutated alleles that are comolimneary to the probe so this one will emit a flurorecence. If test DNA also has the mutated alleles it too will release light so there are 2 lights.
49
What is the polymerase chain reaction?
The polymerase chain reaction is a biomedical technology in molecular biology that can amplify a short length of DNA to thousands more copies, allowing the DNA to be analysed. It is artificial replication of DNA
50
What 4 things does the polymerase chain reaction rely on?
What the PCR relies on:
- DNA is made of two anti pararell backbone strands
- Each strand of DNA has a 5' end and a 3' end
- DNA only grows from the 3' end
51
What are three ways the polymerase chain reaction differs from DNA replication in the body?
PCR vs DNA replicarion:
- in PCR only short sequences can be replicated not whole chromosomes
- PCR requires a DNA primer molecule to make the process start. DNA replication in the biody already has RNA primer/ doesn't need DNA primer
- To separate the DNA strands and bind primers PCR needs heating and cooling but DNA replication uses enzymes
52
How does the polymerase chain reaction differ from when it was first created?
Development of PCR:
- It was first time consuming-- had to heat to denature and cool DNA to 35 degrees to anneal primersa and allow DNA polymerase to work
- Now Taq DNA polymerase is used from a thermophullic bacterium whch is stable at high temperatures so such cooling is not needed
53
List the applications of the polymerase chain reaction
Application of PCR:
- Tissue typing
- Detection of oncogenes
- Detecting mutations
- Identifying viral infections
- Monitoring spread of diseases and detecting emergence of virulent subtypes
- Forensic science
- Research into extinct and extant organisms
54
Briefly outline the stages of Polymerase chain reaction
1. DNA mixed
2. heated to 94-96 degrees to make 2 single strands
3. cooled to 68 degrees making primers anneal making tiny bits of double stranded DNA
3. Taq polymerase binds where double stranded
4. Heated to 72 degrees
5. addition of nucleotides
6. Taq reaches the end of the DNA. A whole new double stranded DNA formed
55
In polymerase chain reaction at what rate does DNA increase?
DNA amount increases exponentially
56
In the first stage of PCR what is added to DNA?
DNA is mixed with DNA nucleotides, primers, Taq DNA polymerase and magnesium ions (a cofactor)
57
Why is the mixture heated to 94-96 degrees in PCR?
2. The mixture is heated to 94-96 degrees to break hydrogen bonds between complimentary nucleotide base pairs, denaturing the DNA into 2 single strands
58
What happens when the mixture in PCR is cooled to 68 degrees?
3. The mixture is cooled to 68 degrees so that primers can anneal by hydrogen bonding to one end of the single strand. This creates a small section of double stranded DNA at the end of each single strand molecule
59
Where does Taq DNA polymerase bind?
4. Taq DNA polymerase can bind to the end where there is a double strand
60
Why is the temperature heated to 72 degrees in PCR?
5. Heated to 72 degrees, keeping the DNA a single strand and this is the Taq DNA polymerase optimum temperature
61
What does Taq DNA polymerase do?
6. Taq DNA polymerase catalyses the addition of DNA nucleotides to the single stranded DNA in the end with the primer in the 5'---3' direction
62
What happens when Taq DNA polymerase reaches the end of the DNA?
7. When the Taq DNA polymerase reaches the end of the DNA a new double strand on DNA has been generated and the process begins again
63
What is tissue typing? PCR
Tissue typing is an application of PCR and is to check donor and recipient tissues prior to transplantation to reduce risk of rejection
64
What is detection of oncogenes? PCR
Oncogene detection. Meaning if the type of mutation involved in a specific patient's cancer can be found medication can be more tailored
65
Examples of detecting mutagens? PCR
Detecting mutagens examples:
- see if parents are carrier
- check fetal cells in prenatal screening
- Cell of embryo analysed before implantation in IVF
66
How do we identify viral infections? PCR
Identify viral infections by detecting a small amount of viral genome amongst the host cell's DNA
67
PCR for monitoring spread of infectious disease?
You can use PCR to monitor the spread of infectious disease and detect emergence of virulent (hostile) subtypes
68
How is PCR used in forensics?
Forensic science with PCR
- small quantities of DNA amplified to identify criminals
- ascertain parentage
69
How is PCR used in research?
PCR in research
- amplifying DNA from extinct animals
- look at what genes are turned on and off in extant organisms
70
What is extant?
Extant means organisms that are still around today
71
What is genetic engineering?
Genetic engineering is taking genes from one organism's DNA and inserting them into another organism DNA
72
Basics of genetic engineering
1. DNA probe finds the gene we need and restriction enzymes cut out the desired gene
2. DNA vector is taken from a bacteria and cut with restriction enzymes
3. gene is placed into the vector with ligase enzymes
4. Transgenic bacteria is crated
5. bacteria multiply
73
What is recombinant DNA?
Recombinant DNA is a composite DNA molecule created in vitro by joining foreign DNA with a vector molecule
74
What are the 4 stages of genetic engineering?
stages:
1. obtain required gene
2. place gene into vector
3. vector placed into recipient cell
4. recipient cell expresses the novel gene
75
How do we obtain the gene for genetic engineering
- DNA probe locates gene and restriction cut it
- design PCR polymers for the gene if they know its sequence
- gene synthesised using automated polynucleotide synthesiser if we know the nucleotide sequence
- obtain mRNA from cells where the gene is expressed then reverse transcriptase catalyses the formation of single strand of complimentary DNA using mRNA as a template. primers and DNA polymerase can make I into a double strand
76
How do we place the gene into a vector using plasmids
plasmids obtained from the bacteria mixed with restriction enzymes so they get cut at special recognition sites leaving sticky ends. Free nucleotides are added to the ends of the gene to be inserted then gene and plasmid anneal with ligase.
77
How do we place genes into a vector using viruses
The gene is sealed into an attenuated virus that could carry it into the host cell
78
What are four ways of getting the vector into the recipient cell?
- electroporation where a high voltage is applied to the cell to disrupt the membrane
- electrofusion where electric fields help to introduce DNA into cells
- Transfection where DNA can be packaged into a bacteriophage which can transfect the host cell
- recombinant plasmids are inserted into the bacterium which infects some plants and naturally inserts into genome into the hoist cells genome
79
How do we directly introduce a gene into a recipient cell?
Small pieces of gold or tungsten are coated with DNA and shot into plant cells by a gene gun
80
What is assaying?
Assaying is checking the plasmid has been taken up and that the gene has been inserted into the plasmid
81
Why do we need to assay
You need to check the plasmid has been taken up and that the insulin is actually in it so that it is not pointless for the bacteria to reproduce so we don't waste time and money
82
How do we do check plasmid has been inserted?
1. bacteria is only ampicillin resistant when the plasmid has been inserted
- bacteria dies on ampicillin = not taken up plasmid
- bacteria lives on ampicillin = taken up plasmid
83
how do we check the insulin gene has actually entered the plasmid?
2. bacteria is tetracycline resistant only without the insulin gene
- bacteria survives on tetracycline = no insulin gene
- bacteria dies on tetracycline = insulin is in the plasmid
84
Stages of making human insulin frim genetically modified bacteria
1. Scientists obtain mRNA from beta cells in the islets of langerhan in the human pancreas where insulin is made
2. reverse transcriptase is added making a single strand of complimentary DNA + DNA polymerase makes it double
3. free unpaired nucleotides added at the ends of DNA makes sticky ends
4. ligase added and insulin gene inserted into plasmid from E.coli (recombinant plasmids)
5. E.coli mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions to make them take up plasmids
6. reproduce
85
Ethical issues of genetic manipulation
- changing the face of landscapes
- vastly different organisms from the wild relatives
- selective breeding is hit or miss
86
Benefits and hazards of using E coli
Benefits
we can make human insulin to treat diabetes and make human growth hormone for pituitary dwarfism
Hazards
Microorganisms could escape into the wild and create antibiotic resistance
87
Beneftis and hazards of GM mice
Benefits
medical research, genes and testing therapies
Hazards
Animal welfare
88
benefits and hazards of pathogens (viruses)
benefits
vaccinations and gene therapy
hazards
Increase risk of cancer or gene regulation goes wrong
89
What is the basic principle of gene therapy?
The basic principle is o insert a functional allele of a particular gene into cells that contain only a mutated and non functioning allele of that gene.
If the inserted allele is expressed then the individual will produce a functioning protein and no longer have the symptoms associated with the genetic disorder
90
What is somatic cell gene therapy?
somatic cell gene therapy only affects certain cells and the alterations made to a patients genome are not passed to their offspring
91
What are the 3 methods of somatic cell gene therapy?
- liposomes
- artificial chromosomes (extra chromosome that coexists with the other 46)
- viruses
92
Hoe do liposomes treat genetic disorders like cystic fibrosis?
Patients with CF lack functioning CFTR gene which is a transmembrane chloride ion channel
1. alleles are packaged in small spheres of lipid bilayer to make liposomes
2. liposomes in aerosol inhaler and sprayed into nose
3. liposomes pass through plasma membrane of cells into respiratory tract
4. liposomes also pass through nuclear envelope and insert into host genome. The host cell expresses CFTR protein
5. Epithelial cells in lining respiratory tract are replaced every 10-14 days so treatment needs to be done at regular intervals
93
Why does liposome aerosol therapy need to be done regularly?
Epithelial cells in lining respiratory tract are replaced every 10-14 days so treatment needs to be done at regular intervals
94
How are viruses used for somatic cell gene therapy?
Viruses from somatic cell gene therapy
- Used as vectors
- Virus that usually infects humans is genetically modified to encase a functioning allele.
- cannot cause disease
- enters the recipient cells genome taking allele with it
95
Problems with using viruses as gene delivery agents
problems with viruses
- viruses may still provoke an immune response or inflammation
- patient may become immune to the virus making later deliveries impossible
- virus may inset allele into a location in the genome hat disrupts a gene involved in regulating cell division increasing cancer risk
- virus may insert the alleles into patients genome in a location that disrupts regulation of expression of other genes
96
What is germ line gene therapy?
- Germ line gene therapy alters genome of gametes or zygotes to alter the patients cells and their offspring
- It can change the genetic makeup of many people without their consent
- issues with where gene is inserted (regulation of -expression or cell division)
- Strict guidelines are needed
97
procedure of DNA profiling
1. DNA obtained from individual via mouth swab, blood, hair, ancient bone
2. DNA digested by restriction enzymes cutting at either sides of the short tandem repeats in fragments
3. fragments are separated by electrophoresis
4. banding pattern created
5. DNA of who the individual is being compared to is treated with the same restriction enzymes and subjected to electrophoresis
6. banding patterns compared
98
What are short tandem repeats?
Short tandem repeats are sequences of DNA that no not code for proteins. Highly variable region short repeating lengths of D A the exact number of STRS varies from person to person
99
How are DNA profiles analysed?
Analysis
- Short tandem repeats
- STR separated by electrophoresis
- 13 analysed simultaneously
- related individuals will have similar banding patterns
100
Discusses STRS, chances of sharing them etc
- chances an individual sharing one STR is 5-20%
- chances of all STR regions being the same ( sharing STR sequences at all loci) in 2 individuals is 1 x 10 to power of 18
- identical twins have the same at all loci
101
3 applications of DNA profiling
- forensic science (Nazi war criminals, victims0
- maternity and paternity disputes
- analysis of diseases (huntingtons and sickle cell anaemia)
102
Applications of gene sequencing list
- human genome project 1990-2003
- genome wide comparisons
- comparison between species
- evolutionary relationships
- variation between individuals
- predicting amino acid sequences of proteins
- synthetic biology
103
Describe predicting amino acid sequence as an application of gene sequencing
If researchers have an organisms genome sequenced and know which genes code for a specific protein, we can use base triplet code knowledge and amino acids to determine primary structure. We can find out which gens code for intron sand which for exons
104
Discuss synthetic biology
Synthetic biology is a science concerned with building and designing useful biological devises and systems.
Wants to build engineered biological systems that can process information, provide food, maintain human health and enhance the environment. The sequences provide potential building blocks
involves: evolutionary biology, biophysics, molecular biology
105
What did the human genome project find?
We have 24,000 genes not 100,000
106
Discuss comparison between species as an application of gene sequencing
- few human genes are unique most have counter parts in other organisms (conserved)
- in evolution genes get co opted to do new tasks
- many differences between organisms are not because they have totally different genes but that some shared genes have been altered and work differently
- changes to regulatory region of dna have altered expression of genomes increasing number of proteins made
107
Discuss evolutionary relationships as an application of gene sequencing
- Comparing genomes of closely related organisms helps confirm their relationship or lea to reclassification
- We can verify evolutionary history of extinct organisms by taking bone and teeth DNA
- bacterial genomes can be extracted from bone to see evolutionary history of pathogens
108
Discuss variation between individuals as an application of gene sequencing
- only 0.1% of our gens is not shared with others and there are specific places where DNA sequences can differ due to random mutations like substitutions
- These substitutions occur at single nucleotide polymorphisms (SNPs)
- some have no effect on protein and some alter proteins or the way RNA regulates the expression of another gene
109
Discuss methylation in variation between individuals
Methylation of certain chemical groups in DNA plays major role in regulating gene expression in eukaryotes.
If we can map out this methylation of whole genomes we can understand development of certain diseases . EPIGENETICS. E.G. why cancer types do/ don't develop in genetically similar individuals
110
Bioethics of synthetic biology
- ethics
- biosecurity
- reward and risk managed
- not about making synthetic life forms
- extensive regulation sin place and many papers written
