Manipulating genomes Flashcards
(98 cards)
1
Q
Sequencing - Why are so many copies of the unknown DNA sequence needed?`
A
To ensure multiple copies of every possible length strand is produced, and no bases are ‘missed’.
2
Q
Seq - What is the function of the primers
A
To give DNA polymerase a double stranded section of DNA to start with - a strand to elongate.
3
Q
Seq - What is the function of the dNTPs
A
To elongate the strand.
4
Q
Seq - What is the function of the ddNTPs?
A
To terminate the strand.
5
Q
Seq - What is the role of electrophoresis in the process
A
To separate the strands according to length - shortest emerges from the process first.
6
Q
Seq - How is the final sequence produced?
A
A laser causes the labelled ddNTP to fluoresce and give out a pulse of coloured light. This is detected and recorded on an electropherogram.
7
Q
Seq - How are very long (»> 1000bp) sections of DNA sequenced
A
- Shotgun sequencing.
- Multiple copies are randomly cut and the fragments sequenced.
- Computers look for overlaps and the fragments are assembled into the original sequence
8
Q
What is a genome?
A
all of the genes (including mitochondrial) - both the coding and non-coding DNA
9
Q
Sanger seq basics?
A
- essentially DNA rep but using a mixture of normal nucleotides that allow the new strand to elongate and a set of chemically altered nucleotides that terminate the elongating chain
- these altered nucleotides are labelled w a fluorescent marker that can be recognised by an automatic seq. machine
10
Q
dNTP?
A
adds to 3’ end of DNA strand, elongating the chain
11
Q
ddNTP?
A
adds to 3’ end, terminating it
12
Q
how does electrophoresis produce an electropherogram?
A
- As the DNA fragments emerge in the capillary tube, a laser causes the markers to fluoresce
- this is picked up by a sensor and an electropherogram is produced showing the base seq
13
Q
Shotgun seq?
A
• sequenced fragments from sanger seq ordered using shotgun sequencing
14
Q
weakness of shotgun seq?
A
unclear where the repeat sequences overlap if repeat seq
15
Q
PCR?
A
Used for making many copies of a specific seq. of DNA
16
Q
What’s needed for PCR?
A
- Original DNA sample - the template
- short DNA primer that are comp to the start and the end of the seq to be amplified - the primers ‘bracket’ the seq
- ddNTPS - all 4
- a thermostable DNA polymerase usually Taq polymerase
- thermal cycler machine
- Eppendorf tubes
17
Q
3 stages of PCR?
A
- Denaturation
- Primer annealing
- Elongation
18
Q
- Denaturation?
A
- 95 degrees c for 30s
* ds DNA is separated into ss by breaking H bonds between the strands
19
Q
- primer annealing?
A
- 55 degrees
* primer bind to the start and end of the DNA template by complementary binding
20
Q
- elongation?
A
- 72 degrees for at least 1 min
- DNAP extends the primers
- works best at 72, it’s the optimum T
- DNAP adds to bases to the primer, building complementary strands of DNA, producing a ds DNA molecule identical to the original seq
21
Q
after elongation?
A
- then step 1 again
* repeating this process results in an exponentially inc the no. of DNA molecules
22
Q
in a diagram the primers would be at ? sides of opp DNA strands?
A
opposite, DNA strands are antiparallel
23
Q
Benefits of PCR?
A
+ detects mutation
+ recombine - gene therapy - attaching DNA to some other genome
+ paternity testing
+ solving crimes
24
Q
Applications of PCR?
A
- start of sanger seq, to make copies of the DNA to be seq
- DNA profiling -many copies of the DNA sample are made before RE used
- making copies of a gene being inserted into a vector for GE
- Amplification of small DNA samples for testing e.g. pre-natal diagnosis
25
Electrophoresis uses?
* ss
* DNA profiling
* can be used to separate proteins too
* separates diff lengths of DNA
26
what moves the furthest in electrophoresis?
* Smallest DNA fragments
* less resisted by gel
* so detected first
27
How does shotgun seq work?
* multipole copies of the genome are fragmented randomly into short, sequencable lengths
* fragments are seq
* then overlapping base sequences are identified using computers
* the fragments can then be put in order and large seq of DNA can be assembled
28
new seq tech?
* inc speed of seq
* brought down costs
* allowed whole genome seq to happen
29
uses of seq?
* find alleles giving predisposition e.g. cancer - preventative treatment
* diagnose genetic conditions - e.g. preimplantation genetic diagnosis
* discover phylogeny/ evolutionary relationships
* adds to sci knowledge/ database of sequences
30
why is Taq polymerase used?
can heat to high temps, other DNA polymerase would denature
31
why is a thermal cycler machine used?
can change T
32
new high throughput techniques?
use nanopores - artificially constructed channel proteins thru which DNA moves, reading its base seq
33
DNA profiling basics?
• everyone has a unique combination of DNA (except identical twins)
• but is more similar to close family members
• so DNA profiling is a useful tool for:
paternity testing, forensics, classification of organisms, pre-disease
34
DNA profiling involves looking at sections of DNA which?
* vary between individuals
* could be STRs
* VNTRs
* specific alleles
35
STRs?
short seq of non-coding DNA that repeat a diff no. of times in diff indivis
36
VNTRs?
* minisatellites/microsatellites/ VNTRs
| * repeating sections of non-coding DNA found in the introns of the genome
37
looking at specific alleles?
specific alleles for specific genes that are known to give e.g. a predisposition can be looked at
38
how are these variable regions found?
* probes (short lengths of radioactively labelled DNA)
* are made that are comp to the seq of bases of interest
* the probes bind to the DNA of interest and show up as bands on a DNA profile
39
DNA profiling uses?
* paternity
* forensics
* other familial relationship testing
* ancestry investigation - e.g. 23 & me
40
DNA profiling is more interested in
* junk - non coding DNA:
* introns
* Telomeres - VNTRs
* centromeres
41
What is a DNA profile?
• producing an image of the patterns of repeats in a person is known as their DNA profile
42
where do RE cut?
restriction sites. They cut DNA strands into the introns at defined points but leaves the STRs in tact.
43
Steps of DNA profiling?
1. extraction - DNA is extracted from cells
2. DNA digested using RE, which cut it at restriction sites
3. DNA fragments are separated using gel electrophoresis.
4. Southern blotting
5. Radioactive probes are added which determine which DNA fragments are seen at the end by binding to spec seq
6. the X ray film can record where the prove has stuck
7. the DNA fingerprint shows where the probe stuck
44
Southern blotting?
The gel separated DNA fragments are now transferred to the nylon membrane, so that the paper now carries an exact replica of the DNA on the gel
45
How is the DNA profile developed?
* If probes = radioactive then x ray
| * if fluorescent, observe under UV light giving a pattern of bars
46
2 types of GE organism?
* Transgenic
| * Cisgenic
47
Transgenic?
- gene came from another organism
48
Cisgenic?
- gene came from the same species
49
GE organisms are often referred to as?
GMOs - anything that has had a change made to is genetic material
50
GE is?
using technology to change the genetic material of an organism
51
GE e.g.?
* gene from eel causes fish to keep growing and producing more meat
* E coli modified to produce human insulin or HGH
52
General principles of GE?
1. gene from donor organism(s) cut out of genome using RE
2. A vector is used to transfer the gene(s) into recipient organism
3. recipient is tested to see whether the transfer has been successful - recombinant DNA has been produced. Organism needs to take DNA in
4. now transgenic/ GM recipient organism synthesises the protein/ proteins coded for by the inserted gene - the gene product
53
Tools needed in GE?
* RE
| * vectors
54
RE?
* Naturally made by bacteria
* cuts at restriction sites
* cuts can leave blunt ends where both strands are the same length
* or sticky ends - one strand is longer than the other, leaving a short length of ss DNA
55
what are restriction sites?
unique base sequences where RE cut
56
Vectors are used to?
* transfer the gene(s) that have been cut from the genome of one organism into the recipient organism
* there are many diff vectors which can be used depending on the type of recipient organism
57
if the RO is a bacterium then
a plasmid/ bacteriophage (virus that infects bacteria) can be used
58
if RO is plant?
agrobacterium vector used
59
if RO is an animal ?
liposome (artificially constructed vesicle) used`
60
Plasmid vectors?
* plasmids often contain genes that code for proteins that give the bacterium resistance to antibi
* these are easily transferred between cells so useful for getting recombinant DNA into bacterial cells
61
Recombinant DNA?
DNA that has genes from >1 organism
62
general principles of using a plasmid vector?
* plasmids cut open using RE
* gene(s) inserted
* plasmid rejoined - now contains the gene
* plasmid inserted into recipient bacterium which now starts to express the gene
63
Using GE E.coli to produce HGH?
1. DNA extracted from human cells and digested with BamH1
2. forms many fragments of diff sizes, one of which contains the HGH gene - this is a known length
3. HGH gene separated from other fragments using gel elec
4. multiple copies made using PCR
5. PB322 plasmids are then extracted from bacteria using enzymes and centrifugation
6. plasmids the cut using BamH1, leaving the same sticky ends as the HGH gene
7. plasmids and copies of the HGH gene are then mixed w DNA ligase which joins the sugar phosphate backs of the DNA molc
64
2 possibilities (E. coli to produce HGH)?
1. HGH gene incorporated into the plasmid and the plasmid is transformed - now a recombinant plasmid
2. plasmid closes up again and is not transformed
65
when bacterial cells are under stress?
they start taking in things from their envir
66
generally, when a plasmid vector is used one of 3 things can happen?
* Bacterial cells take up the transformed plasmid
* Take up untransformed plasmid
* don't take up any plasmid
67
Problem with GE plasmids and bacterial cells?
* can't tell the bacterial cells apart
* so marker genes on the plasmid are used
* mark which cells have taken up the transformed plasmid
* this is called replica plating
68
Replica plating?
* master plate - agar plate containning ampicillin
* the colonies on this have taken up a plasmid - dk if transformed tho
* sterile pad placed onto replica plate, some cells stick to it
* pad placed onto surface of replica plate containing TC
* cells are transferred onto this plate in exactly the same place they were on the master plate
* plate incubated, some colonies develop
* colonies that develop on this plate contain cells with untransformed plasmid - unwanted
* can go back to master plate and see where the colonies are growing there but not on the replica plate - wanted
69
bacteria that have taken ip the gene will show
AMP resistance but not TC resistance
70
any bacteria that have not taken up the gene
will show Ampicillin and tetracycline resistance
71
how can the resistance of these bacteria be tested?
* spread them onto an agar gel plate and incubate
* cells will divide and form colonies on the gel
* each colony consists of 1000s of identical cells because started from a single cell
* Ab added - colonies won't for if now resitant to that AB
72
why do transformed plasmids not have TC resistance?
BamH1 cuts right in the middle of the TC gene destroying it
73
Marker genes?
•used to indicate which cells have taken up the gene of interest
- which cells have been successfully transformed
74
Using GE bacteria to express the gene for insulin ?
* principles the same, but the way the gene is isolated is diff
* comp mRNA + reverse transcriptase + NTPs to make comp DNA
* heat the comp DNA to break H bonds, separating strands
* add more nucleotides to complete comp DNA
* Can use probes to identify the gene
75
How is this diff from cutting the gene from a chromosome using RE?
* When RE used, they cut at a specific base sequence, leaving sticky ends
* then gel elec to isolate gene
* But with this method, because we couldn't identify the gene so insulin mRNA was collected from B cells
* identified and isolated
* reverse transcriptase and NTPs used to make comp strand of DNA
* The synthesised strand is heated to sep strands, DNA P binds to promoter region and free nucleotides are added to elongate the new strand
* the DNA strand is then cut using RE to leave sticky ends which means it can then be inserted into a plasmid
76
what is gene therapy?
Treating genetically inherited conditions with healthy genetic material
77
2 types of GT?
* somatic cell gene therapy
| * germ line gene therapy
78
Somatic cell gene therapy?
• introducing a healthy allele into somatic ells that are affected by a defective allele e.g. CF, haemophilia - all controlled by 1 gene
79
germ line cell therapy?
* cells that / into gametes
* introducing healthy allele into the germ line cells ^, usually egg cell or zygote during IVF
* resulting baby born healthy, grows on to pass allele onto offspring
* alters genome of offspring too
* designer babies
80
- of germ line therapy?
- not allowed rn
- impact of intervention on germ line cells unknown
- done w/o consent and once done process is irreversible so human rights of unborn child violated
- tech may eventually be used to enable ppl to choose desirable characteristics of offspring
81
+ of germ line therapy?
+ development of gene editing technology
| + very precise way of changing small prop. of the genome at a time
82
+ of somatic cell gene therapy?
+ successful treatments have been reported - including retinal disease
83
- of somatic cell gene therapy?
- temp solution for treated individual. Allele will be passed on every time the cell divides by mitosis but somatic cells have a limited life, and are replaced by from stem cells which have the faulty allele
- treated individuals will still pass the faulty allele on to any children
84
Ethics of GMOs? +
+ insect resistant crops reduce need for pesticides, reduced harm to other insects and human health
+ no evidence to suggest that GMOs are unsafe to eat - we eat DNA whenever we eat food and DNA is the same whatever the organism it comes from
+ the risk of genetic pollution is small - GM plants are unlikely to have an + in the wild and are rarely grown near related plans they could pollinate
85
Ethics of GMOs? -
- little research into long term effects of eating GMOs and tech is rel. recent
- GMOs may outcompete natuve species
- Ownership problems. Can a company own a combination of genes that they have assembled in an organism?
- fear of the unknown, and fear of ‘experts’ and their ability to ‘play god’ by ‘tinkering with nature’.
86
E.g. of GMO issues/ techniques
* GM pathogens for research
* insect resistance in GM soya
* Pharming
* patenting and tech transfer
87
GM pathogens for research?
• MO have been GM to produce diff substances e.g. insulin and vaccines
88
Pros of using GM pathogens in research?
* used in research for developing medical treatment and industrial process
* development of gene therapy
* strict reg when pathogens are GM
* used safely for many yrs
89
cons of using GM pathogens in research?
* GM pathogens could be used for biological warfare
| * attempts to modify genome to be more resistant raises ethical concerns
90
Insect resistance in genetically modified soya?
* gene inherited so that soya beans produce the Bt protein
* Bt protein toxic to pest insects, so used as pesticide
* one strain of soya beans has been E to be resistance to weedkiller and contain Bt protein
91
Pros of insect resistance in GM soya?
* reduce amount of pesticide needed - protects envir and helps poor farmers
* inc yield
92
Cons of insect resistance in GM soya?
* non-pest insects and insect - eating predators may be harmed by the toxins in the GM soya
* pests may become resistant to pesticide
93
Pharming?
* genetically modifying animals to produce pharmaceuticals
* aspects: creation of animal model
* addition/ removal of genes so that animals develop certain diseases, acting as models for develop of new therapies
* creating human proteins - the intro of a human gene coding for a medically req proteins
94
pros of pharming?
* medicines produced
* bacteria can't produce all of the complex proteins produced by eukarya
* new therapies can be developed
* more ethical than using a human
95
cons of pharming?
* ethical issues - is it right to put human genes into animals & is it acceptable to put genes from 1 species into another w/o being certain that it wouldn't cause harm
* harder to use animals than plants
96
issues relating to patenting and tech transfer?
* ppl in less developed countries will be prevented from using GMOs due to patents and issues of tech transder
* ppl who need it most may not be able to afford it
* can't use seeds from 1 year to next
97
pros of issues relating to patenting and tech transfer?
* some organisations have developed rice spec. for less economically developed countries
* investment was made so only fair to expect a return
98
cons of issues relating to patenting and tech transfer?
* farmers dis - can't use sseds from one year to the next
| * could have sig. health benefits if not patented
