20 Biotechnology Flashcards
(111 cards)
1
Q
What is recombinant DNA?
A
DNA molecules from from segments of two different sources
2
Q
What is recombinant DNA?
A
DNA molecules from from segments of two different sources
3
Q
What is a common gene cloning occurs is formed? (not virus)
A
With bacterial ‘plasmids’
4
Q
What does ’gene cloning’ refer to?
A
The production of multiple copies of the same gene
5
Q
What is it called when genes are duplicated for research?
A
‘Gene cloning;
6
Q
How is gene cloning performed with plasmids?
A
The plasmid from a bacterium i.e. E coli is extracted.
The gene of interest is taken from another cell and added to the plasmid, forming recombinant DNA.
The plasmid is then returned to the bacterium which divides to form new copies of the plasmid and thus gene of interest.
7
Q
What is a typical bacterium used for gene cloning using plasmids?
A
E. coli
8
Q
What is the most common method to produce recombinant DNA?
A
The use of ‘restriction enzymes’
9
Q
How is bacterial DNA protected from degradation by its own restriction enzymes?
A
By the addition of methyl groups (-CH3) to adenines or cytosines within the sequences recognized by the enzymes.
10
Q
How are restriction enzymes used to produce recombinant DNA?
A
‘Restriction enzymes’ bind to specific base pair sequences called ‘restriction site.’
Within this site they break the DNA at particular points. This forms two ‘restriction fragments.’
Due to the way the DNA was broken the ends probably overlap i.e. ==⎻ and _==. This leaves an unpaired region called the ’sticky end.’
Two restriction fragments from different DNA molecules can combine if their sticky ends are complementary. DNA ligase seals the strands.
11
Q
What does ’restriction site’ refer to?
A
The specific base sequence that a particular restriction enzymes binds to.
12
Q
What is the place that the restriction enzymes bind to called?
A
The restriction site
13
Q
What are the resultant strands of DNA after restriction called?
A
Restriction fragments
14
Q
What is a DNA molecule that can carry foreign DNA into a host cell called?
A
A ‘cloning vector’
15
Q
What is a cloning vector?
A
A DNA molecule that can carry foreign DNA into a host cell where it can replicate.
16
Q
What is special about bacterial plasmids derived from E.coli?
A
They often have genetically engineered amp R and LacZ genes
17
Q
What does ’ampR’ refer to?
A
A gene often added to bacterial plaids to make them resistant to the antibiotic ampicillin.
18
Q
How can it be determined whether bacteria are recombinant? (non-genetic method)
A
The plasmids are engineered so that they have the ampR gene (ampicilin resistance) and the LacZ gene to break down lactose. Conversely the original host bacteria are engineered to not have the LacZ gene and thus can’t break down lactose.
The first step in determining whether the bacteria are recombinant is to dose them with ampicillin. Only those who are resistant could have taken up the plasmid.
Second, the presence of X-gal in the medium allows us to distinguish colonies with recombinant plasmids from those with nonrecombinant plasmids. Colonies containing non- recombinant plasmids have the lacZ gene intact and will produce functional β-galactosidase. These colonies will be blue because the enzyme hydrolyzes the X-gal in the medium, forming a blue product. The others will be white
19
Q
What does X-gal’’ refer to?
A
A substance which when broken down forms a blue product. Otherwise it is white.
Often used to determine if bacteria are recombinant.
20
Q
What is a collection of an organism’s gene in the form of plasmids called?
A
A plasmid library/genomic library
21
Q
What does ’genomic library’ refer to?
A
A collection of all an organism’s DNA in an accessible form i.e. in many plasmids
22
Q
What does ‘plasmid library’ refer to?
A
A collection of all an organism’s DNA in the form of many plasmids
23
Q
What vector is typically used for library construction?
A
Bacterial artificial chromosome (BAC) although plasmids can still be used.
24
Q
What does ’BAC’ refer to?
A
Bacterial artificial chromosome
25
What are Bacterial artificial chromosomes?
A specialised vector in which all but the genes necessary for replication are removed.
26
What is the fundamental different between normal plasmids and BAC?
In plasmids 90% of the DNA is normal non-recombinat DNA.
However in BACs the genes carried are 1000 times larger than the vector itself.
27
Besides Plasmid libraries, what is a common way of collecting all of an organism’s DNA?
With cDNA (complementary DNA) libraries
28
What does ‘cDNA’ stand for?
Complementary DNA
29
What is cDNA?
DNA which is formed from mRNA. It is therefore complementary to the RNA and thus identical to the original DNA
30
What is the fundamental way cDNA libraries differ from plasmid libraries?
Plasmid libraries contain the entirety of an organism’s DNA.
Conversely cDNA is contracted from mRNA and thus contains only the expressed genes. This means that is does not include promoter regions etc., silenced genes or introns.
31
How is cDNA formed?
Reverse transcrip- tase is added to a test tube containing mRNA isolated from a certain type of cell.
Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT’s (lots of Thymine) as a DNA primer.
mRNA is degraded by another enzyme.
DNA polymerase synthesizes the second strand, using a primer in the reaction mixture.
Thus cDNA is formed without introns.
32
What is a common method for detecting a specific sequence of DNA?
Nucleic acid hybridisation
33
What is nucleic acid hybridisation used for?
Detecting a specific sequence of DNA
34
How does nucleic acid hybridisation work?
The DNA wanting to be screened is stored in ‘multiwell plates’ i.e. those things we used when doing food tests or mixing paints. cells form each are transferred onto specific places on a nylon membrane.
’Nucleic acid probes’ are synthesised that are complementary for the sequence to be found. They are then tagged with radiation etc.
The nucleic acid probes are then added to the multi well plates so that they bind to complementary sequences. Any excess are washed away.
Photographic plate is then placed over the multi wells. Any regions where the probes are present will cause dark spots on the film, indicating that the desired sequence is at that location.
35
What is a common issue when adding eukaryotic genes to bacteria? How can this be solved
The eukaryotic genes often have promoter regions that are are not recognised but the bacteria and thus not expressed.
This is solved with ‘expression vectors'
36
What are expression vectors?
A cloning vector that has a highly active bacterial promoter upstream of the restriction site so that the downstream eukaryotic gene is still expressed.
37
How are recombinant plasmids formed?
With restriction enzymes.
38
Why is the use of cDNA beneficial?
- It shortens the gene segment and thus allows more to be placed in one vector
- Prokaryotes do not posses the capability to remove introns. Therefore the cDNA form, which lacks introns, is used when RNA splicing must occur for the gene to function.
39
What are the basic ways DNA is added to a cells besides with plasmids?
Electroporation and with Agrobacterium or with tiny needles
40
What is a electroporation?
A electric pulse is applied to cells to create temporary holes in the plasma membrane through which DNA can enter and hopefully enter the genome
41
In what organisms can electroporation be performed?
Animals and bacteria
42
In what species is Agrobacterium used to add DNA?
Plants only
43
What is Agrobacterium?
Agrobacterius tumiferens is a soil bacteria (rhizobacterium)
44
What is a common gene cloning occurs is formed? (not virus)
With bacterial ‘plasmids'
45
What does ’gene cloning’ refer to?
The production of multiple copies of the same gene
46
What is it called when genes are duplicated for research?
'Gene cloning;
47
How is gene cloning performed with plasmids?
The plasmid from a bacterium i.e. E coli is extracted.
The gene of interest is taken from another cell and added to the plasmid, forming recombinant DNA.
The plasmid is then returned to the bacterium which divides to form new copies of the plasmid and thus gene of interest.
48
What is a typical bacterium used for gene cloning using plasmids?
E. coli
49
What is the most common method to produce recombinant DNA?
The use of ‘restriction enzymes'
50
How is bacterial DNA protected from degradation by its own restriction enzymes?
By the addition of methyl groups (-CH3) to adenines or cytosines within the sequences recognized by the enzymes.
51
How are restriction enzymes used to produce recombinant DNA?
‘Restriction enzymes’ bind to specific base pair sequences called ‘restriction site.’
Within this site they break the DNA at particular points. This forms two ‘restriction fragments.’
Due to the way the DNA was broken the ends probably overlap i.e. ==⎻ and _==. This leaves an unpaired region called the ’sticky end.’
Two restriction fragments from different DNA molecules can combine if their sticky ends are complementary. DNA ligase seals the strands.
52
What does ’restriction site’ refer to?
The specific base sequence that a particular restriction enzymes binds to.
53
What is the place that the restriction enzymes bind to called?
The restriction site
54
What are the resultant strands of DNA after restriction called?
Restriction fragments
55
What is a DNA molecule that can carry foreign DNA into a host cell called?
A ‘cloning vector'
56
What is a cloning vector?
A DNA molecule that can carry foreign DNA into a host cell where it can replicate.
57
What is special about bacterial plasmids derived from E.coli?
They often have genetically engineered amp R and LacZ genes
58
What does ’ampR’ refer to?
A gene often added to bacterial plaids to make them resistant to the antibiotic ampicillin.
59
How can it be determined whether bacteria are recombinant? (non-genetic method)
The plasmids are engineered so that they have the ampR gene (ampicilin resistance) and the LacZ gene to break down lactose. Conversely the original host bacteria are engineered to not have the LacZ gene and thus can’t break down lactose.
The first step in determining whether the bacteria are recombinant is to dose them with ampicillin. Only those who are resistant could have taken up the plasmid.
Second, the presence of X-gal in the medium allows us to distinguish colonies with recombinant plasmids from those with nonrecombinant plasmids. Colonies containing non- recombinant plasmids have the lacZ gene intact and will produce functional β-galactosidase. These colonies will be blue because the enzyme hydrolyzes the X-gal in the medium, forming a blue product. The others will be white
60
What does X-gal’’ refer to?
A substance which when broken down forms a blue product. Otherwise it is white.
Often used to determine if bacteria are recombinant.
61
What is a collection of an organism’s gene in the form of plasmids called?
A plasmid library/genomic library
62
What does ’genomic library’ refer to?
A collection of all an organism’s DNA in an accessible form i.e. in many plasmids
63
What does ‘plasmid library’ refer to?
A collection of all an organism’s DNA in the form of many plasmids
64
What vector is typically used for library construction?
Bacterial artificial chromosome (BAC) although plasmids can still be used.
65
What does ’BAC’ refer to?
Bacterial artificial chromosome
66
What are Bacterial artificial chromosomes?
A specialised vector in which all but the genes necessary for replication are removed.
67
What is the fundamental different between normal plasmids and BAC?
In plasmids 90% of the DNA is normal non-recombinat DNA.
However in BACs the genes carried are 1000 times larger than the vector itself.
68
Besides Plasmid libraries, what is a common way of collecting all of an organism’s DNA?
With cDNA (complementary DNA) libraries
69
What does ‘cDNA’ stand for?
Complementary DNA
70
What is cDNA?
DNA which is formed from mRNA. It is therefore complementary to the RNA and thus identical to the original DNA
71
What is the fundamental way cDNA libraries differ from plasmid libraries?
Plasmid libraries contain the entirety of an organism’s DNA.
Conversely cDNA is contracted from mRNA and thus contains only the expressed genes. This means that is does not include promoter regions etc., silenced genes or introns.
72
How is cDNA formed?
Reverse transcrip- tase is added to a test tube containing mRNA isolated from a certain type of cell.
Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT’s (lots of Thymine) as a DNA primer.
mRNA is degraded by another enzyme.
DNA polymerase synthesizes the second strand, using a primer in the reaction mixture.
Thus cDNA is formed without introns.
73
What is a common method for detecting a specific sequence of DNA?
Nucleic acid hybridisation
74
What is nucleic acid hybridisation used for?
Detecting a specific sequence of DNA
75
How does nucleic acid hybridisation work?
The DNA wanting to be screened is stored in ‘multiwell plates’ i.e. those things we used when doing food tests or mixing paints. cells form each are transferred onto specific places on a nylon membrane.
’Nucleic acid probes’ are synthesised that are complementary for the sequence to be found. They are then tagged with radiation etc.
The nucleic acid probes are then added to the multi well plates so that they bind to complementary sequences. Any excess are washed away.
Photographic plate is then placed over the multi wells. Any regions where the probes are present will cause dark spots on the film, indicating that the desired sequence is at that location.
76
What is a common issue when adding eukaryotic genes to bacteria? How can this be solved
The eukaryotic genes often have promoter regions that are are not recognised but the bacteria and thus not expressed.
This is solved with ‘expression vectors'
77
What are expression vectors?
A cloning vector that has a highly active bacterial promoter upstream of the restriction site so that the downstream eukaryotic gene is still expressed.
78
How are recombinant plasmids formed?
With restriction enzymes.
79
Why is the use of cDNA beneficial?
- It shortens the gene segment and thus allows more to be placed in one vector
- Prokaryotes do not posses the capability to remove introns. Therefore the cDNA form, which lacks introns, is used when RNA splicing must occur for the gene to function.
80
What are the basic ways DNA is added to a cells besides with plasmids?
Electroporation and with Agrobacterium or with tiny needles
81
What is a electroporation?
A electric pulse is applied to cells to create temporary holes in the plasma membrane through which DNA can enter and hopefully enter the genome
82
In what organisms can electroporation be performed?
Animals and bacteria
83
In what species is Agrobacterium used to add DNA?
Plants only
84
What is Agrobacterium?
Agrobacterius tumiferens is a soil bacteria (rhizobacterium)
85
Is BAC used for amplifying DNA in vitro or in vivo?
In vivo (in living organism)
86
What is the most common way to amplify DNA in vitro?
Polymerase Chain Reaction
87
Is PCR used in vitro or in vivo?
In vitro
88
What does PCR stand for?
Polymerase Chain Reaction.
89
What are the steps of PCR?
Denaturation: Heat briefly separates DNA strands. Similar in effect to helicase.
Primers are added for the desired region. One primer is complementary to one end of the target sequence on one strand; the second primer is complementary to the other end of the sequence on the other strand.
Annealing: The DNA is cooled to allow primers to form hydrogen bonds with ends of target sequence.
Extension: DNA polymerase adds nucleotides to the 3’ end of each primer.
The cycle is repeated to yield more molecules
90
What is an advantage of PCR?
It can work with small quantities of DNA in vitro
91
How many molecules does PCR form?
It doubles the number of molecules per cycle.
However variation in the way primers bind i.e. sometimes only one binds means that ofter cycle 3 8 molecules are formed but only 2 match the original sequence.
Thus the amount of DNA doubles every 3 cycles.
92
What are some disadvantages of PCR?
Occasional errors during PCR replication impose limits on the number of good copies that can be made by this method.
When PCR is used to provide the specific DNA fragment for cloning, the resulting clones are sequenced to select clones with error-free inserts.
PCR errors also impose limits on the length of DNA fragments that can be copied.
93
What is unique about the enzymes used in PCR? Where do they originate?
Due to the denaturation (hot) then annealing the polymerase must be able to withstand high heat.
Therefore Taq polymerase from hot springs bacteria is used
94
What does ’Taq polymerase’ refer to?
A form of polymerase derived from hot springs bacteria and thus having a high enough denaturation point to be used in PCR.
95
What does electrophoresis study in basic?
The size of various fragments of DNA
96
What is the gel used for electrophoresis typically composed of?
Agarose (a polysaccharide)
97
What is agarose?
A polysaccharide often used in electrophoresis.
98
How does electrophoresis work?
The fragments wanting to be tested are placed in a gel containing agarose.
An electric charge is ran though the plate. Because nucleic acid molecules carry negative charges on their phosphate groups, they all travel toward the positive pole on the opposite end.
As they move, the agarose fibers impedes longer molecules more than it does shorter ones, separating them by length.
Thus the shorter the bands are, the farther they will travel.
A DNA binding dye such as ethidium bromide is added. Under UV light is fluoresces to reveal the bands
99
What is a common DNA dye used in electrophoresis?
Ethidum bromide
100
What is ethidium bromide?
A DNA bindding dye which is added to samples after electrophoresis.
It is then washed away then exposed to UV. Any bands that fluoresce contain DNA.
101
Besides analysis, what can electrophoresis be used for?
DNA can be recovered undamaged from gels, the procedure also provides a way to prepare pure samples of individual fragments.
102
What is electrophoresis typically used to analyse?
Directly it measures fragment length.
Therefore it can be used to investigate changes in restriction sites.
103
What does ’polymorphism’ refer to?
Variations in DNA sequences among a population.
104
What is analysis using electrophoresis to deduce how the restriction sites vary called?
'Restriction fragment length polymorphism’ (RFLP)
105
What is RFLP?
Restriction fragment length polymorphism
106
What is ‘Restriction fragment length polymorphism’?
A method of comparing the differences between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites.
Electrophoresis is used to determine how long the various fragments are and thus where the restriction sites are.
107
What is the basic purpose of Southern Blotting?
To determine whether a sample of DNA contains a desired base sequence.
108
What method could be used to determine if a sample of DNA had a specific base sequence?
Southern Blot
109
What are the steps for performing a Southern Blot?
Preparation of restriction fragments:
Each DNA sample is mixed with the same restriction enzyme to yield mixture of many restriction fragments.
Gel electrophoresis: The restriction fragments in each sample are separated by electrophoresis bands. However since a large section of DNA was used the identification of individual bands is practically impossible - it appears as a smear
DNA transfer (blotting): A sponge is placed in a bath of alkaline solution. The gel is placed on tope, then a nitro-cellulose membrane and finally a heavy weight. Capillary action pulls the alkaline solution upward through the gel, denaturing and transferring the DNA to a nitrocellulose membrane. This produces a blot with a pattern of DNA bands exactly like that of the gel.
Hybridization with labeled probe: The nitrocellulose blot is exposed to a probe labeled in some way. Probe molecules attach by base-pairing to any restriction fragments containing the desired based sequence
A sheet of photographic film is laid over the blot. The radioactivity in the probe causes dark spots where the tagged DNA is present.
110
What fundamental methods does Southern Blotting combine?
Electrophoresis and Nucleic Acid Hybridisation
111
Why is it important that Southern Blotting uses both Electrophoresis and Nucleic Acid Hybridisation.
Electrophoresis could not be used on its own as the large segment of DNA would form so many bands that only a smear would be present.
Nucleic acids hybridisation on its own would only indicate the presence or absence of a bee sequence.
However by combining these methods it can be determined where the base sequence occurs in a specific gene.
