6.3 Manipulating Genomes Flashcards
1
Q
Define polymerase chain reaction
A
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
2
Q
Applications of PCR
A
Tissue typing Detection of oncogenes Detecting mutations Identifying viral infections Monitoring the spread of infectious disease Forensic science Research
3
Q
Applications of PCR
A
Tissue typing Detection of oncogenes Detecting mutations Identifying viral infections Monitoring the spread of infectious disease Forensic science Research
4
Q
Define DNA ligase
A
Enzymes that catalyses the joining of sugar and phosphate groups writhing DNA
5
Q
Define electroporation
A
Method for introducing a vector with a novel gene into a cell; a pulse of electricity makes the recipient cell membrane more porous
6
Q
Define plasmids
A
Small loops of DNA in prokaryotic cells
7
Q
Define recombinant DNA
A
A composite DNA molecule created in vitro by joining foreign DNA with a vector molecule such as a plasmid
8
Q
Define restriction enzymes
A
Endonuclease enzymes that cleave DNA molecules at recognition sites
9
Q
Define vector
A
In gene technology, anything that can carry/ insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria
10
Q
Necessary stages of genetic engineering
A
- required gene is obtained
- copy of the gene is placed inside a vector
- vector carries gene to recipient cell
- the recipient expresses the novel gene
11
Q
What is DNA sequencing?
A
A technique that allows genes to be isolated and read
12
Q
Who developed the technique that allows you to sequence whole genomes?
A
Fred Sanger
13
Q
What was sangers approach?
A
Use a single strand of DNA as a template for four experiments in different dishes
14
Q
What was in each one of Sanger’s dishes?
A
Solution with four bases and DNA polymerase
15
Q
What did Sanger add to each tube?
A
Modified version of one of the DNA bases that meant once it was added to complementary DNA, no more bases could be added
16
Q
What is each base labelled with in Sanger’s experiment?
A
Radioactive isotope
17
Q
In gel electrophoresis, which fragments travel the furthest?
A
Smaller ones
18
Q
What were the terminal bases labelled with in the first DNA sequencing machine?
A
Fluorescent dyes which glowed when scanned with a laser beam
19
Q
What does pyrosequencing involve?
A
Synthesising a single strand of DNA, complementary to the one being sequences, one base at a time whilst detecting by light emission which base was added at each step
20
Q
Steps of pyrosequencing
A
-DNA is cut into 300-800 base fragments using a nebuliser
-fragments degraded to single stranded DNA
-they are immobilised
-primer added and DNA incubated with enzymes
-activated nucleotides added and
light detected
21
Q
In pyrosequencing, what enzymes is the DNA incubated with?
A
DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin
22
Q
What activated nucleotides are used in pyrosequencing?
A
ATP
TTP
CTP
GTP
23
Q
What happens when an activated nucleotide is added in pyrosequencing?
A
- Two extra phosphoryls are released as pyrophosphate (PPi)
- The enzyme ATP sulfurylase converts pyrophosphate to ATP by combining it with APS
- ATP and luciferin are converted to oxyluciferin by luciferase
- this generates light
24
Q
What are unincorporated nucleotides degraded by?
A
Apyrase
25
What are the applications of gene sequencing?
- genome wide comparisons between individuals
- genome wide comparisons between species
- sequences of amino acids in polypeptides to be predicted
- development of synthetic biology
26
Examples of synthetic biology applications?
```
Information storage
Production of medicines
Novel proteins
Biosensors
Nanotechnology
```
27
Examples of nanotechnology
Amyloid fibres making biofilms
| For functions such as adhesion
28
Basic stages of DNA profiling
Extract sample of DNA
DNA digested with restriction enzymes, which cut into fragments
Fragments separated by gel electrophoresis and stained
Banding pattern
Compare with other DNA
29
Why is restriction fragment length polymorphism analysis no longer used?
Laborious
30
What types of DNA are analysed in DNA profiling?
Short tandem repeat (STR) sequences of DNA
31
How many STRs are analysed simultaneously?
13
32
Applications of DNA profiling
Forensic science
Maternity and paternity disputes
Analysis of disease
Plant and animal breeding (reduces inbreeding)
33
How has profiling been used in forensic science?
Identifying nazi war criminals in South America
Identify remains of Romanov family
Victims bodies in disasters
34
How much of STR repeats come from mother and father?
Half from mother half from father
35
Example of how DNA profiling is used in disease analysis
- detect haemoglobin present and aid diagnosis of sickle cell anaemia
- Huntington’s repeat sequence number
36
What is PCR?
A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
37
What does PCR do?
Amplify amount of DNA
38
What facts does PCR rely on?
- DNA is anti parallel
- have 5’ and 3’ end
- DNA only grows from 3’ end
- complementary bases pair up
39
How does PCR differ from DNA replication?
Short sequence replicated, not chromosomes
Requires primer molecules
Cycle of heating and cooling need to separate DNA strands, bind primers to strands and for strands to be replicated
40
What type of reaction is PCR?
Cyclic
41
Why was the process of PCR slow at first and how was it sped up?
- DNA was heated to denature it and then cooled to 35 degreees to anneal primers and allow polymerase to work
- DNA polymerase was obtained from Thermophilus aquaticus, thermophilic bacterium
- Taq polymerase is stable at high temperatures
42
Applications of PCR?
- tissue typing
- detection of oncogenes
- detecting mutations
- identifying viral infections
- ANALYSIS OF DISEASE RISK
- FORENSICS
- research
- to produce enough DNA to mix with cut plasmids to insert into bacteria (in vivo cloning)
43
What is tissue typing?
Donor and recipient tissues typed before transplantation to reduce risk of rejection
44
What viral infections can PCR identify?
HIV or hepatitis C
45
What’s the sample of DNA mixed with in PCR to start? + Where is this mixture put?
```
DNA nucleotides
Primers
Magnesium ions
Taq DNA polymerase
-All in thermocycler
```
46
What is the DNA heated to the first time in PCR and why?
94-96 degrees
To break H bonds between complementary nucleotide bases
Two single strands of DNA
47
What is the PCR mixture cooled to and why and what is produced?
68 degrees
Primers can anneal to one end of each single strand of DNA
A small section of double stranded DNA at the end of each single stranded molecule
48
What is the optimum temp for Taq polymerase?
72 degrees and the mixture is raised to this
49
What does Taq polymerase do?
Catalyses the added of nucleotides to single stranded DNA
Start at the end with primer
5’ to 3’
50
How does the amount of DNA increase in PCR?
Exponentially
| 1-2-4-8-16-32-64-128
51
After running gel electrophoresis on your DNA sequence what do you expose it to?
Photographic paper due to the radioactive isotopes
52
Name of sequences forensic DNA tests use to identify individuals
Variable number tandem repeats
53
Advantages of PCR?
- rapid
| - no living things involved
54
Disadvantages of PCR?
Contaminant DNA may also be amplified
55
What is electrophoresis?
process used to separate proteins or DNA fragments of different sizes
56
What does electrophoresis use?
Agarose gel plate covered by a buffer solution
| Electrodes at each end
57
What is the overall charge of DNA and why?
Negative due to its many phosphate groups
58
Which way do DNA fragments move in gel electrophoresis?
Towards the positive electrode (anode)
59
Why are the samples in electrophoresis only separated by size?
Because different size fragments of DNA all have a similar surface charge
60
Which fragments of DNA move the furthest in gel electrophoresis?
The shortest ones
61
Difference between electrophoresis of proteins and electrophoresis of DNA?
-proteins = often carried out in the presence of a charged detergent such as sodium dodecyl sulfate (SDS) which equalises surface charge
62
Why do we need SDS in protein electrophoresis?
Because proteins may have different surface charge
63
What can gel electrophoresis of proteins be used to analyse?
types of haemoglobin for:
- sickle cell anaemia
- aplastic anaemia, thalassaemia and leukaemia
64
What are DNA probes?
Small bits of DNA which know sequences
single stranded
tagges
65
What can DNA probes be labelled using?
- radioactive marker
| - fluorescent marker (emits colour change on exposure to UV light)
66
What can we use DNA probes for?
- locating a gene to be used in genetic engineering
- genome comparison studies
- identifying alleles for genetic disease
67
What is a DNA microarray?
A number of different probes on a fixed surface
68
How do microarrays work?
You put your sample of DNA onto the plate with the probes and scan it
- probes may be flourescently labelled and fluoresced when hybridised to samples of added DNA
- tell you which probes were successful in detecting complementary sequences
- can infer which sequences of DNA were in samples
69
Use of microarrays and electrophoresis
- locating and identifying alleles
- genetic screening
- personalised medicine
- genetic counselling
70
Define DNA ligase
enzyme that catalyses the joining of sugar and phosphate groups within DNA
71
What is electroporation?
method for introducinga vector with a novel gene into a cell; a pulse of electricity makes the recipient cell more porous
72
What are plasmids?
small loops of DNA in prokaryotic cells
73
What is recombiant DNA?
a composite DNA molecule created in vitro by joining foreign DNA with vector molecule such as a plasmid
74
What are restriction enzymes?
endonuclease enzymes that cleave DNA molecules at specific recognition sites
75
What is a vector?
in gene technology, anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria
76
Necessary stages of genetic engineering
- required gene obtained
- copy placed into vector
- vector carried gene into recipient cell
- recipient expresses the novel gene
77
Methods for obtaining required gene in genetic engineering
- mRNA obtained, reverse transcriptase makes a single strand of complementary DNA (cDNA) with the addition of primers and DNA polymerase can make double stranded
- automated polynucleotide synthesiser (if you know whole sequence)
- PCR to amplify known gene
- DNA probe locates gene and cut out using restriction enzymes
78
How can you place a gene into a vector?
- plasmids obtained from microorganisms and mix with restriction enzymes that cut plasmid at specific recognition sites
- the plasmid now has exposed unpaired nucleotide bases, sticky ends
- free nucleotide bases that match are added to the gene bing inserted and DNA ligase catalyses the annealing of the gene into plasmid
- gene may be seal into a weakened virus that could carry it into a host cell
79
Methods of putting vector into recipient cell
-heat shock treatment
-electroporation
-electrofusion
-transfection
T1 (recombinant) plasmids inserted into bacterium, Agrobacterium tumefaciens, which naturally inserts
-gene gun
80
What is heat shock treatment?
bacteria subjects to alternating 0-42 degrees in the presence of calcium chloride
membranes become more porous, DNA can enter
81
What is electroporation?
high voltage pulse disrupts membrane
82
What is electrofusion?
electrical fields help introduce DNA into cells
83
What is transfection?
DNA packaged into bacteriophage
| transfects host cell
84
What are direct methods of introducing genes into recipient?
If plants are not susceptible to A.tumefaciens
small pieces of gold or tungsten coated with DNA and shot into plant cells
GENE GUN
85
Ethical considerations of genetic engineering?
- escape of resistance genes (into weeds, eg herbicide resistance)
- health and welfare of GM animals (testing)
86
Positive of GM soya
resistant to herbicides
| weeds competing with soya plants can be killed using herbicides
87
Negative of GM soya
herbicide resistance pass into weeds producing superweeds
88
Positive of GM pathogens
- viruses, modified to having no virulence, make vaccines that don't make people ill
- used as vectors in gene therapy
89
Negative of GM pathogens
-uses of viruses in gene therapy, allele may be inserted into genome in a way that increases risk of cancer or interferes with gene regulation
90
Positive of GM animals to produce pharmaceuticals
-gene inserted into goats or sheep and they produce human protein in their milk
91
Negative of GM animals to produce pharmaceuticals
welfare of GM goats and sheep
| however, valuable and well looked after
92
Issues relating to patenting and technology transfer
making genetically modified seed available to poor farmers
93
difference between somatic and germ line gene therapy?
somatic can target specific tissues in need of treatment
| germline cannot
