8.4 Gene technologies Flashcards
1
Q
What is recombinant DNA technology
A
The transfer of fragments of DNA from one organism, or species, to another
2
Q
What is the name of the organism that receives the fragment of DNA
A
Recipient
3
Q
What 2 reasons mean the recipient can translate the DNA within the cell
A
- Genetic code is universal
- Transcription and translation mechanisms are universal
4
Q
What is the recipient organism said to be after receiving the DNA fragments
A
Transgenic
5
Q
What 2 fields may recombinant DNA technologies be beneficial
A
- Medicine
- Agriculture
6
Q
Define donor DNA
A
Gene that is isolated for insertion
7
Q
Define plasmids
A
Circular loops of DNA can be used as vector
8
Q
What is meant by the word vector in terms of recombinant DNA technology
A
Carries the DNA into recipient
9
Q
Define restriction endonucleases
A
Enzymes that cut DNA at specific restriction sites
10
Q
Define DNA ligases
A
Enzymes that join sections of DNA together
11
Q
Define sticky ends
A
2 ends of ‘cut’ DNA segments that have unpaired
12
Q
Define recombinant DNA
A
DNA which is formed when a piece of foreign DNA is incorporated into the plasmid from a bacterium
13
Q
Define reverse transciptase
A
Enzymes used to synthesise DNA from mRNA in specific cells
14
Q
Define clone
A
A population of genetically identical cells or organisms
15
Q
What happens in the isolation part of the DNA technology
A
Isolating DNA fragment that contains the gene of desired protein
16
Q
What are the 3 steps when isolating the desired gene
A
- Locate the gene, in cells that naturally produce the protein
- Identify gene locus using a gene probe
- Isolate gene from DNA
17
Q
What organism does restriction endonucleases naturally occur in
A
Bacteria
18
Q
What are the names of the sites where the restriction endonucleases cut the DNA
A
At specific restriction sites/ recognition sites
19
Q
What bonds do restriction endonucleases hydrolyse
A
Phosphodiester bonds
20
Q
Recognition sites are always _________
A
Palindromic
21
Q
What does palindromic mean
A
Read the same forward and backward
22
Q
What do restriction endonucleases do
A
Cut double-stranded DNA into fragments
23
Q
What are the steps involved in using reverses transcriptase to isolate the desired gene
A
- Extract mRNA that codes for the gene from a cell that naturally produces the protein
- Mix the mRNA with reverse transcriptase, so complementary DNA (cDNA) is formed
- Add the single-stranded DNA strand with DNA polymerase
- A double-stranded DNA will form
24
Q
What are the advantages of using reverse transcriptase to isolate genes compared to restriction endonucleases
A
- There are far more mRNA molecules that carry the desired gene compared to DNA
- mRNA is in the cytoplasm whereas DNA is in the nucleoplasm so fewer membrane to pass
- No introns in mRNA, so can be put into bacteria cells
25
Why is it beneficial to have no introns in the gene that's transferred into bacteria
Since bacteria have no introns so don't know what to do with it
26
What is needed for the gene machine to work
The primary structure of the protein
27
Why does the primary structure of the protein need to be known to use the gene machine
- Identify the amino acid sequence
- Then determine the mRNA codons
- Then determine the complementary DNA triplets
- The determine the base sequence needed for the gene machine
28
What does the gene machine produce
Short DNA fragments
29
What is the name of the short DNA fragments that the gene machine produces
Oligo-nucleotides
30
What is the name of the enzyme that joins the oligo-nucleotide together in the gene machine
DNA ligase
31
What are the benefits of the gene machine
- Intron free
- Most accurate
- Fastest
32
Where the DNA of 2 different organisms is combined, the product is known as
Recombinant
33
One method of producing DNA fragments is to make DNA from RNA using an enzyme called
Reverse transcriptase
34
Once restriction endonucleases have cut the DNA, it leaves 2 types of ends, name the 2 types
- Sticky ends
- Blunt ends
35
During the insertion process of gene technology, what is inserted into one end of the gene which enables transcription factors to bind
Promoter region
36
During the insertion process of gene technology, what is inserted into the other end of the gene which releases RNA polymerase to end transcription
Terminator region
37
When inserting gene into vector, what is important about the restriction endonucleases that are used
They must be the same, the same enzyme that cut the donor DNA into small fragments must cut the plasmid too
38
Why must the vector and the donor DNA be cut with the same restriction endonucleases
So the sticky ends are complementary on the gene and the plasmid
39
What is the role of DNA ligase in insertion process
Splice the gene into the vector
40
When using a plasmid as a vector, what may happen after the plasmid has been cut open
The plasmid may bind to itself again without the donor DNA within
41
How are plasmids reintroduced
By mixing plasmids with bacteria cells, so bacteria can take plasmids up
42
What is the general term for the things that are used to identify bacteria with genes
Genetic markers
43
What are genetic markers
Genes that are easily identifiable
44
What are the 3 main genetic markers
- Genes that result in antibiotic resistance
- Genes that code for fluorescent proteins
- Genes that code for enzymes whose action can be identified
45
How is the donor DNA spliced into a plasmid with antibiotic resistance to be used as a genetic marker
Cut the plasmid with the 2 resistant genes, in the centre of one of the resistant genes and insert the fragment of donor DNA inside.
46
How does the donor DNA being inserted into resistant region on a plasmid act as a genetic marker
- The bacteria that has the recombinant plasmid won't be resistant to the antibiotic where the donor DNA was added to
47
What are the 3 possible option of bacteria after plasmid have had fragments of DNA added and mixed with the bacteria
- Bacteria with no plasmids
- Bacteria with recombinant plasmids
- Bacteria with original plasmids
48
How can scientists identify which bacteria has which type of plasmid
Replica plating
49
Where is the gene inserted in the vector, when fluorescent markers are used
In the middle of the GFP gene
50
Are bacteria with recombinant plasmid that have fluorescent markers, fluorescent or not
Not fluorescent
51
What does lactase do
Turn colourless substrate of lactose into a blue product
52
How are enzyme markers used to identify bacteria with recombinant plasmids
Genes are added to the centre of lactase, so bacteria with recombinant plasmids are white
53
What happens in the culturing process of DNA technology
- Remove the identified bacteria
- Add to tank for fermentation
- Where the bacteria replicate by binary fission which produces genetically identical bacteria all containing recombinant plasmids
54
What does in vivo mean
Inside of living organism
55
What does in vitro mean
Outside of living organism
56
What is the method when cloning DNA in vitro
PCR machine
57
What is the method when cloning DNA in vivo
Using bacteria cells to clone DNA
58
What does PCR stand for
Polymerase chain reaction
59
What is the role of PCR
DNA amplification
60
What does the PCR machine do to the number of DNA molecules
Increase the number exponentially (double the number per unit of time)
61
What is added to the PCR machine (4 things)
- DNA nucleotides
- The gene
- Primers
- DNA polymerase
62
What type of DNA polymerase is used in the PCR machine
Taq
63
What are primers
Short single-stranded DNA
64
What are the roles of primers in PCR machine (2 roles)
Primers are complementary to the bases at the start of the gene we wish to clone, and primers signal DNA polymerase to start synthesising bonds
65
After everything is added to the PCR machine, what temperature is the machine heated to
95 degrees celsius
66
Why is the PCR machine heated once everything is added to the machine
High temperature breaks the hydrogen bonds, which unzips the DNA, so both strands can act as template
67
After the PCR machine is heated to 95 degrees, what temperature is the machine then cooled to
55 degrees celsius
68
Why is the PCR machine cooled to 55 degrees
Since this is the temperature that the primers complementary base pair (anneal) to DNA strand
69
What is the biological word for when primers complementary base pair with DNA strand
Anneal
70
What is the advantage of primers annealing with DNA strand
To stop DNA from rejoining together
71
After the PCR machine is cooled, what temperature is the machine then heated too
72 degrees celsius
72
Why is the PCR machine heated to 72 degrees
Since this is the optimum temperature for Taq DNA polymerase
73
What are the 5 limitations of PCR
- Contamination to solution
- Error rate
- Size of DNA fragment that is copied
- PCR is sensitive to inhibitors
- Limit to amplification
74
Why is contamination a limitation to PCR
Any DNA in machine is amplified
75
Why is error rate a limitation to PCR
Taq DNA polymerase cannot proof read mutation in DNA replication so errors accumulate
76
Why is size of DNA fragments a limitation to PCR
PCR machine cannot read anymore that 3000 bases
77
Why is limit to amplification a limitation to PCR
- Exponential growth only occurs for roughly 20 cycles, since enzymes begin to denature, concentration of nucleotides decrease
78
What is the comparative statement:
In vivo cloning culturing techniques are required
In vitro cloning no culturing techniques are required
79
What is the comparative statement:
In vivo cloning once in bacteria the DNA will automatically be copied
In vitro cloning, requires correct primers to be present for copying to occur
80
What is the comparative statement:
In vivo cloning there's almost no risk of contamination
In vitro cloning any contaminated DNA will also be amplified
81
What is the comparative statement:
In vivo cloning DNA polymerase has 'proof reading function'
In vitro cloning DNA polymerase does not have 'proof reading function'
82
What is the comparative statement:
Vivo cloning is very accurate
Vitro cloning errors accumulate
83
What is the comparative statement:
In vivo cloning transformed bacteria can synthesise gene product
In vitro cloning genes must be inserted into cells to synthesise gene products
84
What is the comparative statement:
Vivo cloning is relatively slow
Vitro cloning is extremely rapid
85
What is the comparative statement:
In vivo cloning specific genes are isolated
In vitro cloning whole or broken down DNA can be copied
86
What is the comparative statement:
In vivo cloning copying can be effective up to 3 million base pairs
In vitro cloning copying can be effective up to 3000 base pairs
87
What are the medical benefits of recombinant DNA technology
- Synthesis of medical products e.g. insulin
- Using genetically modified bacteria in vaccines
88
What are the argicultural benefits of recombinant DNA technology
- Enhancing crop growth
- Genetically modify crops to aid survival e.g. drought and disease resistant
89
What is a DNA probe
Short segments of single-stranded DNA
90
What are the 2 methods of labelling DNA probes
- Radioactive labelling
- Fluorescent labelling
91
Why are DNA probes labelled
To identify the probes
92
What are the 2 uses of DNA probes
- Used to locate specific alleles of genes
- Screen patients for hereditary diseases, drug responses, and health risks
93
When using DNA probes to locate mutant alleles, what is the first step
Determine the base sequence of the mutant allele of the gene
94
When using DNA probes, after the base sequence of the mutant alleles has been determined, what then happens
DNA fragments that has complementary base sequence for the mutant allele needs to be produced
95
When using DNA probes, what happens to the fragment of DNA with the complementary base sequence to the mutant allele
DNA fragment is labelled either fluorescently or radioactively, forming the DNA probe
96
What is formed when the DNA fragment is labelled
DNA probe
97
Once the DNA probe has been formed, what happens to it
DNA probe is amplified by the PCR
98
After DNA probe amplification, what happens to the DNA probe
Added to single-stranded patient DNA
99
When will the DNA probe anneal to the patient DNA
When the mutant allele is present
100
What happens to the patients DNA if the mutant allele is present
DNA is labelled by the probe
101
What is used to expose the mutant allele when the DNA probe was labelled fluorescently
UV light
102
Define anneal
When sections of DNA complementary base pair opposed to each individual base
103
Why can transferred DNA be translated within the cells of a transgenic organism (2 reasons)
- Transcription and translation mechanisms are the same in most organisms
- Genetic code is universal
104
Explain what is added to a DNA fragment before it can be transferred to a host cell
- Promotor region - so transcription factors can bind which activates RNA polymerase
- Terminator region- so RNA polymerase is released
105
How is genetic screening carried out
Add multiple DNA probes to a glass slide and then add the patients DNA
106
What is genetic screening used for
Identify genetic disorders
107
What is personalised medicine
When health advice and medicine are given to an individual base on their specific genotype
108
What are the benefits of personalised medicines
- Allows for more effective treatment
- Avoids over-prescribing
109
What is genetic counselling
When advice is given following screening of a disorder
110
What are 2 arguments for genetic screening
- Increase screening/ testing for the disorder e.g. mammograms for breast cancer
- Individuals can make different lifestyle choices
111
What are 2 arguments against genetic screening
- Even if the gene is present, it's not certain the individual will get the disease - so this leads to unnecessary anxiety and stress
- Individuals without the gene may still get the disorder as they aren't just hereditary
112
What does genetic fingerprinting do
Analyse VNTR fragments
113
What does VNTR stand for
Variable number tandem repeats
114
What are the 4 main uses of genetic fingerprinting
- Forensic science at crime scenes
- Paternity test
- Medical diagnosis
- Determine relatedness for breeding
115
More closely related individuals have the more _______ the VNTRs
Similar
116
What is the first step in gel electrophoresis
DNA has to be extracted from biological material
117
Once the DNA has been extracted, what happens to it before is can be used in gel electrophoresis
Cut into fragments using restriction endonucleases
118
Why are DNA fragments different lengths before going into gel electrophoresis
Since recognition sites on DNA are randomly spaced and the restriction endonucleases only cut at the recognition sites
119
After the DNA has been cut into fragments, what then happens in gel electrophoresis
DNA fragments are added into wells in the agar plate and a voltage is applied across the gel
120
In gel electrophoresis, does the DNA move towards the positive or negative end
Towards positive end
121
Why does the DNA more towards the positive end during gel electrophoresis
Since the phosphate group gives DNA a negative charge
122
Do shorter or longer fragments of DNA travel faster and further through the gel
Shorter fragments
123
After the DNA fragments have had time to move through the gel, what is then added to the gel
Alkali
124
What is the role of alkali when added to the gel in gel electrophoresis
Separates the double-stranded DNA into single-stranded DNA
125
Why must the DNA fragments be transferred to nylon membrane instead of staying on the agar gel
As the agar gel dries it shrinks and cracks
126
How are the DNA fragments transferred from gel to nylon membrane
The agar is covered with the nylon membrane and a weight is added on top, the membrane picks up the DNA from each position
127
What is added to the nylon membrane
DNA probes, with complementary base pairs to the VNTRs
128
What must happen after DNA probes are added to the nylon membrane
Wash off any unbound probes
129
The VNTRs in a child come from where
Both the mother and the father
130
What are the pros of genetic fingerprinting
- Non-invasive
- Easy to do and easy to obtain a sample
- Samples that are too small for blood testing can be used
- Used to reverse wrongful convictions
131
What are the cons of genetic fingerprinting
- Requires tight regulation of access
- Violation of privacy
- Stored on a database which can be misused or hacked
- Lack in understanding in reading genetic fingerprinting
- Profiles offer probabilities not absolutes
- Can still result in wrongful convictions
