21: Recombinant DNA Tech Flashcards
1
Q
What is recombinant DNA?
A
DNA of two different organisms that has been combined
2
Q
What is an organism with recombinant DNA inserted called?
A
Transgenic
Genetically modified organism (GMO)
3
Q
Why can recombinant DNA be inserted into an organism?
A
Genetic code is universal
4
Q
What is the process of making a protein using DNA technology of gene transfer?
A
Isolation Insertion Transformation Identification Growth/cloning
5
Q
What occurs in isolation?
A
DNA fragments for desired gene are isolated
6
Q
What occurs in insertion?
A
DNA fragment collected is inserted into a vector
7
Q
What occurs in transformation?
A
Transfer of DNA into suitable host cells
8
Q
What occurs in identification?
A
Host cells which have taken up the gene is identified using gene markers
9
Q
What occurs in growth/cloning?
A
The host cells population is replicated/cloned to produce many copies
10
Q
What are the methods of producing DNA fragments?
A
Conversion of mRNA to cDNA using reverse transcriptase
Restriction endonucleases used to cut fragments containing desired gene from DNA
Creating a gene in the gene machine, from a known protein structure
11
Q
What is cDNA?
A
Complementary DNA
As made of nucleotides which are complementary to the mRNA found
12
Q
What is the process of using reverse transcriptase to make DNA fragments?
A
Cell which produces the protein is selected as have large amounts of relevant mRNA
Reverse transcriptase converts mRNA to ds cDNA
cDNA made to be ss by hydrolysis with an enzyme
DNA polymerase enzyme builds complementary nucleotides on cDNA template
dsDNA is the required gene
13
Q
What are restriction endonucleases?
A
Enzymes found in bacteria which cut up viral DNA
Viruses inject DNA into bacteria to take over cell
14
Q
What is the difference between different restriction endonucleases?
A
Each one cuts a dsDNA at a specific base sequence
15
Q
What is a recognition sequence?
A
Specific base sequence of DNA at which a restriction endonuclease cuts
16
Q
What are blunt ends of DNA?
A
Restriction endonuclease cuts DNA between two opposite base pairs
Leaves two straight edges called blunt ends
17
Q
What are sticky ends of DNA?
A
Restriction endonuclease cuts DNA in a staggered fashion
Leaves an uneven cut in which each strand of DNA has exposed, unpaired bases
18
Q
What is a palindromic sequence?
A
The unpaired bases read from left to right is the same as right to left
19
Q
What is the gene machine?
A
A piece of equipment which manufactures genes in a laboratory
20
Q
How does a gene machine work?
A
Desired nucleotide sequence of genes determined from desired protein
Desired sequence is fed into the computer
Sequence checked for biosafety and biosecurity to keep to international and ethical standards
Computer designs series of oligonucleotides which can be assembled into a desired genes
Automated process, each of oligonucleotides assembled one nucleotide at a time
Oligonucleotides joined together to make a gene, replicated using PCR
21
Q
What is an oligonucleotide?
A
Small, overlapping single strands of nucleotides
22
Q
What is the feature of the gene made from a gene machine?
A
No introns or other non-coding DNA
23
Q
What is done with genes made from the gene machine?
A
Sticky ends used to insert gene into a bacterial plasmid (vector)
Then can be used to store, clone or transfer to different organism
24
Q
What are the advantages of the gene machine?
A
Any nucleotide sequence can be produced in a short time with great accuracy
Artificial genes are free of introns so can be transcribed & translated by prokaryotic cells
25
What is a vector?
Carrying unit for the DNA fragment
| E.g. bacterial plasmid
26
How can cloning of a gene be done?
In vivo - transfer to a host cell using a vector
| In vitro - using PCR
27
How are sticky ends used in vectors?
Exposed bases made from sticky ends
Complementary base pairing occurs between this and DNA from another source cut with same restriction endonuclease
DNA ligase joins the two sections
28
What does DNA ligase do?
Forms the sugar-phosphate backbone between the two different sections of DNA
29
What is a promoter?
Region of DNA which transcription factors and RNA polymerase binds to, allows transcription to occur
30
What must be done to a DNA fragment before it is inserted?
Promoter and terminator region has to be added to the DNA fragment
31
Why do promoter and terminator regions be added to DNA fragment?
Ensures that the DNA is transcribed and made when it is inserted into a living organism
32
What is the most commonly used vector?
Plasmid
33
What are plasmids?
Circular lengths of DNA found in bacteria
Separate from main bacterial DNA
Contain genes from antibiotic resistance
34
Why is the restriction endonuclease used for the DNA the same as the one used for the plasmid?
Ensures sticky ends of opened-up plasmid are complementary to sticky ends of DNA fragment
35
What is transformation?
Process of plasmid being inserted into the bacterial cells
36
What is the process of transformation?
Plasmids and bacteria mixed in medium with Ca2+
Ca2+ and changes to temp makes the bacterial membrane permeable
Plasmids move into the cytoplasm through the membrane
37
Why do all of the bacterial cells not have the DNA fragments?
V few bacterial cells take up plasmids when mixed
Some plasmids will have closed up again without incorporating the DNA fragment
Sometimes DNA fragment ends join back together to form own plasmid
38
How many genes can a plasmid carry?
Many genes for resistance
| One plasmid can dictate resistance from multiple antibiotics
39
What is a marker gene?
A gene used to determine if a fragment has been successfully inserted into an organism's DNA
40
What are some examples of marker genes?
Antibiotic-resistant genes
Fluorescent protein producing gene
Enzyme producing gene whose action can be identified
41
Is antibiotic-resistant marker genes used?
Old technology
| Superseded by other methods
42
How is resistance for ampicillin used to find which bacterial cells have taken up the plasmid?
All bacteria cells grown on medium with ampicillin
Bacterial cells with plasmids will be resistant to ampicillin
Bacterial cells break down ampicillin
Others do not take up plasmid and therefore die
43
What is replica plating?
Process used to find those cells with plasmids that have taken up the new gene
44
What is the process of replica plating?
Filter paper/ felt used to obtain an exact copy of the colony locations on the original plate
Used to transfer to plate containing antibiotic (ampicillin) which all with plasmids have
Those that are alive are used to transfer colonies to plate with different antibiotic (tetracycline) which the fragment would be inserted into the middle of
Those which die have the correct plasmid, those which live have the unaltered plasmid
Replica plating means colony with recombinant DNA can be found on ampicillin plate
45
What is a fluorescent marker?
Gene which produce a protein which is fluorescent
| GFP - green fluorescent protein found in the jellyfish
46
How are fluorescent markers used?
Desired gene transplanted into centre of GFP gene
Plasmid also contains antibiotic resistant gene
Bacterial cells with desired gene will not fluoresce but will survive, and hence can be isolated
47
Why are fluorescent markers used?
Results easily obtainable
No need for replica plating
Therefore more rapid
48
What is an example of an enzyme marker?
Gene that produces lactase
| Lactase turns a specific substrate from colourless to blue
49
What is the polymerase chain reaction (PCR)?
Method of copying fragments of DNA
| Automated process
50
What are the advantages of PCR?
Automated so rapid and efficient
51
What is required for PCR?
```
DNA fragment (to be copied)
DNA polymerase (taq polymerase)
Primers
Nucleotides
Thermocycler
```
52
Why is taq polymerase used for PCR?
Obtained from bacteria in hot environments
| Needed as required to be tolerant to heat and not denature at high temps in the PCR process
53
What is a primer?
Short sequences of nucleotides that have bases complementary to those at one end of each of the two DNA fragments
Prevents two separate strands reforming in PCR
54
What is a thermocycler?
Computer-controlled machine that varies temperatures precisely over a period of time
55
What are the three stages of PCR?
Separation of DNA strands
Addition (annealing) of primers
Synthesis of DNA
56
What occurs in the first stage of PCR?
DNA fragment, primers and taq polymerase placed in thermocycler
Temp increased to 95C, causes two strands of DNA fragment to separate due to H-bonds breaking
57
What occurs in the second stage of PCR?
Mixture cooled to 55C
Causes primers to join to complementary bases at end of DNA fragment
Primers also prevent two separate strands reforming
58
What occurs in the third stage of PCR?
Temp increased to 72C, optimum temp of taq polymerase
Adds complementary nucleotides along each of separated DNA strands
Begins at primer on both strands and adds until the end of the chain
59
What is the function of the primers?
Primers provide sequence for taq polymerase to start DNA copying
Attaches nucleotides to ends of an existing chain
Prevents two separate strands reforming
60
How many copies are made from 1 original fragment?
2 strands (copies) made from each strand present
61
How can you calculate the number of strands found after a number of rounds of PCR?
Number of strands = 2^(number of rounds)
62
Roughly how long does each round of PCR take?
2 minutes
| Fast process
63
What are some uses of PCR?
Provides multiple copies of DNA fragment
| Forensic detection
64
What are the advantages of in vitro gene cloning?
Very rapid
| Does not require living cells
65
What is a disadvantage of using PCR for forensic detection?
Increases amount of contaminating DNA found at the crime scene
66
What are the advantages of in vivo gene cloning?
Useful when introducing gene into another organism
Involves almost no risk of contamination
Very accurate
Cuts out specific genes
Produces transformed bacteria that can be used to produce large quantities of gene products
67
Why is there no risk of contamination with in vivo cloning?
Same restriction endonuclease used to match sticky ends of opened-up plasmids
Contaminant DNA not taken up by plasmid
68
Why is in vivo gene cloning very accurate?
Few errors
| Errors could arise from mutation but it is very rare
69
Why is in vitro gene cloning not accurate?
At one time the PCR process would result in a large number of errors
70
Why is it important in vivo cloning produces transformed bacteria?
Transformed bacteria can produce proteins for commercial or medical use
71
What is a DNA probe?
Short ssDNA that has some kind of label attached that makes it easily identifiable
72
Why are DNA probes and hybridisation used?
To find DNA sequence location responsible for a genetic disease
73
What are the two most commonly used probes?
Radioactively labelled probes
| Fluorescently labelled probes
74
What is a radioactively labelled probe used?
Nucleotides with the isotope 32P
| Identified using X-ray film exposed by radioactivity
75
What is a fluorescently labelled probe?
Emits light under certain conditions
| E.g. probe bound to the target DNA sequence
76
How are DNA probes used to identify particular alleles of genes?
DNA probe has complementary base sequences to DNA with allele of gene wanted
dsDNA is treated to separate the two strands
Separated DNA strands mixed with probe, probe complementary binds to strand in DNA hybridisation
Site at which probe binds is identified by radioactivity or fluorescence that probe emits
77
What is DNA hybridisation?
DNA hybridisation takes place when a section of DNA or RNA combined with ssDNA section which has complementary bases
78
What occurs in DNA hybridisation?
DNA heated to separate
Cooling causes complementary bases on each strand to combine with other complementary sections of DNA present in the mixture
79
What is the process of locating specific alleles of genes?
Determine sequence of nucleotide bases of allele wanted
Fragment of DNA produced which has complementary bases to allele trying to be located
DNA fragment replicated using PCR
DNA probe made by attaching marker to fragment
DNA with suspected mutant allele heated to separate
Cooled in mix with DNA probes
DNA probe binds complementary to allele if present
DNA washed clean of unattached probes
Probe is searched for and if present it is located
80
What occurs if there is a gene mutation to a dominant allele?
All individuals with the allele will have the genetic disorder
81
What occurs if there is a gene mutation to a recessive allele?
Only homozygous recessive people will display the symptoms
82
What is genetic screening?
Testing individuals who may carry a mutant allele to tell if they have a genetic disorder
83
What is personalised medicine?
Genes can affect the effectiveness of some drugs
| With genetic info the doctors can prescribe the drug which will work most effectively
84
What is genetic counselling?
Advice given to people about the genetic diseases they have
| Could be found from family history
85
What does genetic fingerprinting rely upon?
DNA of every individual, except identical twins, is unique
86
What are variable number tandem repeats (VNTRs)?
DNA bases which are non-coding
| Number and lengths of VNTRs is unique per person
87
What is the probability of people having the same VNTRs?
Extremely small
88
How are VNTRs related?
More closely related the individuals, the more similar the VNTRs
89
What is gel electrophoresis used to do?
Separate DNA fragments based on their length
90
How long can the DNA be in gel electrophoresis?
Up to 500 bases long
| Larger genes must be cut into fragments by restriction endonucleases
91
What can be done to DNA in gel electrophoresis to find their relative position on the gel?
Radioactive DNA probes
| Fluorescent probe
92
What is the set up of gel electrophoresis?
Agar gel on plate
| Anode (+) and cathode (-) placed on either side of plate
93
Why do fragments of DNA move in gel electrophoresis?
DNA moves to anode (+)
| Due to -ve phosphate groups
94
What dictates how far DNA moves in gel electrophoresis?
Length of DNA
| Resistance of gel means the larger the fragment the more slowly they move
95
How far will a large DNA fragment move compared to a small one?
Large DNA fragment move less than smaller one
96
What are the 5 main stages of making a genetic fingerprint?
```
Extraction
Digestion
Separation
Hybridisation
Development
```
97
What occurs in the 1st stage of making a genetic fingerprint?
Extraction
DNA is separated from tissue
Amount of DNA increased by PCR
98
What occurs in the 2nd stage of making a genetic fingerprint?
Digestion
DNA cut into fragments using the same restriction endonuclease
Endonuclease chosen which cuts close to, but not within, target DNA
99
What occurs in the 3rd stage of making a genetic fingerprint?
Separation
Gel electrophoresis separates DNA fragments based on size using voltage
Nylon membrane on gel plate
Gel immersed in alkali to separate ds to ss
100
What occurs in the 4th stage of making a genetic fingerprint?
Hybridisation
Radioactive or fluorescent DNA probe used to bind to VNTRs
Probes have complementary base sequence to VNTRs and bind at certain temp and pH
Carried out with different probes for different DNA sequences
101
What occurs in the 5th stage of making a genetic fingerprint?
Development
X-ray film and radiation or UV light source put over nylon membrane
Reveals series of separated DNA fragments as bands
Pattern of bands is unique to every individual except identical twins
102
How are genetic fingerprints compared?
Visually
Then using a scanning machine
Odds calculated of another person having identical fingerprint
103
How does a scanning machine work for genetic fingerprints?
Calculates DNA length fragments from bands
| Uses data by measuring distance travelled in gel electrophoresis
104
What are the uses of genetic fingerprinting?
Genetic relationships and variability
Forensic science
Medical diagnosis
Plant and animal breeding
105
How is DNA fingerprinting used for paternity tests?
Child gets 1/2 from mother and father
| Each band should correspond to either maternal or paternal bands
106
How is DNA fingerprinting used for determining genetic variability in a population?
More closely they are related the closer their genetic fingerprints
Population with all similar have little genetic diversity
107
How is genetic fingerprinting used for forensic science?
Used to determine whether a person was at the crime scene (probability)
Does not meant that person committed the crime
108
What is the probability calculation for genetic fingerprinting based on?
Assumes DNA producing bands is randomly distributed in the community
109
How is genetic fingerprinting used for medical diagnosis?
Allows for diagnosis of genetically inherited diseases if the mutation is known
Identifies nature of microbial infection by comparing microbe fingerprint with patients
110
What is the mutation found in Huntington's disease?
AGC on chromosome 4 is repeated over and over again
| If there over 38 repeats the person is likely to have the disease
111
How is genetic fingerprinting used in plant and animal breeding?
Prevent undesirable inbreeding in farms or zoos
Identifies plants or animals desirable genes for selective breeding
Or determining pedigree
