Restriction Endonucleases and Recombinant DNA Technology. Flashcards
1
Q
Define a bacteriophage?
A
Viruses that affect bacteria.
2
Q
Define a clone?
A
A group of cells that contain the exact same recombinant DNA sequence.
3
Q
Define a cloning vector?
A
A DNA molecule that is capable of replicating inside a host cell.
4
Q
Define competent cells?
A
Cells that are capable of having DNA added to their genome.
5
Q
Define contigs?
A
DNA strands that have been cleaved to produce overlapping sequences.
I.e. they have not been cleaved at all of their restriction sites.
6
Q
Define COS sites?
A
These are cohesion sites and they are another name for sticky ends.
7
Q
Define an endonuclease?
A
Enzymes that cut phosphodiester bonds in the middle of a DNA molecule.
8
Q
Define an exonuclease?
A
Enzymes that cut phosphodiester bonds at the ends of a DNA molecule.
9
Q
Define a nuclease?
A
An enzyme that cleaves phosphodiester bonds in a nucleotide chain.
10
Q
Define an oligonucleotide?
A
A polynucleotide whose molecules contain a small number of molecules.
11
Q
Define phage 𝞬?
A
A particular bacteriophage.
12
Q
Define phage induction?
A
The process that bacteriophages use to integrate their genome into a bacterial cells genome.
13
Q
Define DNA recombinant technology?
A
When DNA sequences from many sources are bought together synthetically to create a new sequence of DNA that is not found naturally.
14
Q
What molecules does DNA recombinant technology heavily rely on?
A
The use of recombinant DNA molecules.
15
Q
Define restriction endonuclease’s?
A
Enzymes that recognise specific base pair sequences and cleave the DNA at that sequence.
16
Q
Define a restriction site?
A
The site on DNA where endonuclease enzymes cleave the 2 strands.
17
Q
What sequences of DNA usually make up restriction sites?
A
Short palindromic sequences.
18
Q
Define the process of screening?
A
The process of searching for a specific DNA sequence in a DNA library.
19
Q
Define a DNA vector?
A
A piece of DNA that is capable of independent growth.
20
Q
DNA vectors are often what kinds of DNA?
A
Bacterial plasmids or viral phages.
21
Q
Cloning the ability to manipulate what?
A
DNA that is then capable of reproducing itself.
22
Q
How are bacterial plasmids used for recombinant processes?
A
By inserting plasmids into bacteria, this gets the genetic information in the plasmids translated into proteins.
E.g. insulin manufacture.
23
Q
A plasmids self replicating?
A
Yes, they will divide every time binary fission occurs.
24
Q
Can plasmids be intergrated into a bacterial genome?
A
Yes.
25
How do bacteriophages infect bacteria cells?
By injecting their viral genome into the cell.
26
How does the bacterial cell make viral proteins?
Viral DNA codes for the host cell machinery to make viral proteins that can be assembled into more viruses.
27
What shape is the DNA that is injected into bacteria by bacteriophages?
It is linear and contains a 5 prime and a 3 prime overhang at each end.
28
What are the 2 overhangs on a linear strand of viral DNA used for after it has been injected into a bacterial cell?
They are complimentary and form bonds to form a piece of DNA that is circular in shape.
29
How does the circular viral DNA incorporate itself into the viral genome?
It bimnds one of its own sequences of DNA to a complimentary sequence on the bacterial DNA.
30
What is the complimentary sequence between the viral DNA and the bacterial DNA known as?
The ATT site.
31
What happens once the ATT site on viral DNA has bound to the bacterial DNA?
The ATT site opens up and allows the viral DNA to become integrated to the bacterial genome.
32
What did scientists use to study bacteriophage's?
They took an E.coli cell and infected it with a lambda phage.
The lambda phage replicated and eventually lysed the cell, releasing a load of baby viruses.
33
What happened when scientists were studying bacteriophage's after the lambda phage had lysed the bacterial cell?
The baby viruses were used to infect K-12 cells.
34
What were the baby viruses called after the lambda phage had lysed the bacterial cell when scientists were studying bacteriophage's?
Lambda-C viruses.
35
What happened during the study of bacteriophage's after the lamda-C viruses had been used to infect K-12 cells?
The viral process only occurred in very rare cases, meaning that the K-12 cells were mainly unharmed.
36
Why did the viral process not occur in the K-12 cells during the study of bacteriophage's?
The K-12 cells could recognise and destroy foreign DNA.
37
How could the K-12 cells recognise foreign DNA during the study of bacteriophage's?
Their DNA is methylated, allowing them to differentiate between their own DNA and foreign unmethylated DNA.
38
How did lambda-C viruses infect some of the K-12 cells during the study of bacteriophage's?
They modified their own DNA by methylating it.
This meant that the K-12 cell could no longer tell the difference between its own DNA and the phage DNA.
39
What enzymes do K-12 bacteria contain that helps them to distinguish between their own DNA and foreign DNA?
Endonuclease's and methylase’s.
A methylase will methylate restriction sites.
The endonuclease will recognise these restriction sites and will cleave them if they are not methylated.
40
When does the methyl-transferase in a K-12 bacterial cell methylate bacteria?
After a restriction enzyme called ECO-R1 has cleaved the bacterial DNA at a specific site.
41
What are the sites in K-12 bacterial DNA that are cleaved by the restriction enzyme ECO-R1 so they can be methylated?
Palindromic sequences.
These sequences read the same on each strand.
42
What kind of methylase will methylate the palindromic sequences on K-12 bacteria?
An ECO-R1 methylase.
43
What is the enzyme that will re-attach the 2 bacterial strands after methylation has occurred in K-12 bacteria?
ECO-R1.
44
Why will palindromic sequences re-attach easily?
Because they form sticky ends.
45
How does the endonuclease in a K-12 bacterial cell protect the bacteria cell against viral invasion?
It scans the DNA within the genome and cleaves any un-methylated sequences.
46
What kind of viral DNA will not be recognised by the endonuclease in the K-12 bacterial cell?
When a virus modifies its own DNA to give the same methylation pattern that is on the host DNA.
47
The process that bacteria use to protect their DNA through methylation is known as what?
As bacterial restriction and modification.
48
Which scientists discovered bacterial restriction and modification?
Meselson and Yuan.
49
How did Meselson and Yuan discover bacterial restriction and modification?
They purified a bacterial enzyme that cleaved lambda-C phage DNA into reproducible pieces.
50
What did Meselson and Yuan name the bacterial enzyme that cleaved lambda-C phage DNA into reproducible pieces
A restriction endonuclease.
51
How does a restriction endonculease in bacteria recongnise bacterial DNA?
Via methylation, which bacteria do in a species specific manner.
52
How do the daughter strands of bacterial DNA become methylated after cell division?
After division, the parent strand remains methylated and the daughter strand will quickly become methylated.
53
What kind of molecules are restriction endonuclease enzyme?
Dimers.
54
What are restriction endonuclease enzymes often used as tools for?
For cloning and other recombinant techniques.
55
What are the 3 major types of restriction endonuclease?
Type 1.
Type 2.
Type 3.
56
How common are type-1 endonculease's?
Less common than the type 2 endonuclease and more common than the type 3.
57
How do type-1 endonculease's cut DNA strands?
They cut both DNA strands at a random location which is far from the recognition site.
58
Are type-1 endonculease's often used in recombinant technology?
No.
59
What are the most common types of endonculease?
Type-2.
60
What are the most frequently used endonuclease's in recombinant technology?
Type-2.
61
How do type-2 endonculease's cut DNA strands?
They cut both DNA strands at specific locations which are usually palindromic and around 4-8 base pairs long.
62
Will type-2 endonculease's create sticky ends when they cut the DNA strands?
Yes.
63
What are the rarest types of endonculease?
Type-3.
64
Are type-3 endonculease's often used in recombinant technology?
No.
65
How do type-3 endonculease's cut DNA strands?
They cut a single DNA strand that is around 25 base pairs downstream of the recognition site.
66
Why are type-1 and type-3 endonuclease's rarely used in recombinant technology?
As they are said to be promiscuous, meaning that they cause random cleavage patterns.
67
Which endonulcease's are said to be the basic tools of cloning?
The type 2’, as they will only cleave at specific sites.
68
How is a restriction endonuclease named?
The first 3 letters are named after the organism that they were found in.
The 4th letter represents the strain of endonuclease.
The letters are followed by a number to indicate the order of discovery of endonuclease's in that organism.
E.g. ECO-R1 was found in E.coli and it was the first restriction endonuclease to be found in E.coli.
69
What do the recognition sites for type-2 endonculease's tend to be?
Around 6 base pairs and they are palindromic.
70
How will a palindromic sequence read on each strand?
They will read exactly the same in the 5 to 3 direction on both strands.
71
What was does an endonuclease read a DNA strand?
In the 5 to 3 direction.
72
What is the most common feature on a DNA strand that has been spliced by a type 2 endonuclease?
To create sticky ends, where there is an overhang on each strand.
73
Why do type II endonuclease's often create sticky ends on each strand?
Because the overhangs are complimentary menaing that the 2 strands will get back together easily.
74
Describe what happens when blunt ends are formed by a restrioction endonuclease?
Blunt ends are created when neither strand has an overhang and these strands are very difficult to re-join.
75
How do researchers predict the probability of getting the right nucleotides for a recognition sequence that reads 5-CCGG-3?
The probability would be 1/4 that the 1st nucleotide is a C.
This multiplied by 1/4 that the 2nd nucleotide is a C.
This is multiplied by 1/4 that the 3rd nucleotide is a G.
This is multiplied by 1/4 that the 4th nucleotide is a G.
Probability of a 5-CCGG-3 match occurring is 1/4 * 1/4 * 1/4 * 1/4 = 1/256.
This means that a recognition site of 4 base pairs is likely to occur every 256 base pairs.
76
How does the amount of base pairs in a recognition site affect the number of nucleotides in between each site?
The more nucleotides in the recognition site means more base pairs between each site.
77
Which enzyme joins the nucleotides together after they have been cut by a restriction endonculease?
DNA ligase catalyses the formation of a phosphodiester bond so that nucleotides can be joined together.
78
How does DNA ligase form a phosphodiester bond between the 2 nucleotides?
By joining the 5-prime phosphate on one nucleotide with a 3-prime hydroxyl group on another.
79
What kind of ends will DNA ligase find easier to bind together?
DNA that has sticky ends or large overhangs rather than with DNA strands that have blunt ends.
80
Is DNA able to splice together DNA from different organisms?
Yes.
As long as both strands have sticky ends.
81
Why are DNA strand with blunt ends difficult for DNA ligase to join back together?
As there are no complimentary overhangs.
82
What enzymes will scientists add to a blunt ends to makle them more likely to join together?
2 enzymes called dTTP and terminal de-oxynucleotidyl transferase are added to blunt end.
2 enzymes called dATP and de-oxynucleotidyl transferase are added to the other blunt end.
83
How do the 4 enzymes that are added to blunt ends make them more likely to join back together?
The dTTP enzyme adds thymine residues to the the 5 prime end of its strand.
The dATP enzyme adds adenine residues to the the 5 prime end of its strand.
These A’s and T’s are complimentary to each other and can be joined together by a DNA ligase.
84
How is a foreign piece of DNA inserted into a restriction site?
DNA ligase can add any piece of DNA to a restriction site as long as their sticky ends match up.
85
What are the basic steps of inserting aforeign piece of DNA into a restriction site?
Open the restriction site using a restriciton endoculease.
Remove the foreign DNA using the same endonculease.
Use DNA ligase to insert the foreign DNA by matching the sticky ends.
86
How must the foreign DNA be instered into the bacterial DNA?
In the correct orientation so that the DNA can be read in the correct direction.
87
What item will help to make sure that the correct orientation is achieved?
Restriction maps.
88
What does restriction mapping help scientists to do?
To create maps of cleavage sites within a DNA molecule.
89
What is a restriction map a description of?
All the locations on a gene that restriction enzymes can bind to.
90
What will restriction endonuclease's do when they come into contact with a DNA molecule?
They will cut the DNA molecule into different segments which are made up of different amounts of base pairs.
91
Why will scientists often add 2 different restriction enzymes to a DNA strand?
To divide it into different segments that are made up of different amounts of base pairs.
92
How are the base pairs that are cleaved by restriction endonuclease's sorted into an order of size?
Via gel electrophoresis.
93
How do scientists figure out which fragments of a DNA strand were cut by a 2nd enzyme in a DNA strand of 200 BP if enzyme 1 cuts 2 segments of 20 and 60 base pairs?
Total DNA strand = 200 base pairs.
Enzyme 1 = 2 segments of 20 and 60 base pairs.
200-80 = 120.
Therefore the segements cut by enzyme 2 will add up to 120.
94
What is step 1 of molecular cloning?
The desired DNA fragments are created by digesting DNA with restriction endonucleases.
95
What is step 2 of molecular cloning after the desired DNA fragmnets have been created?
The cloning vector that the desired DNA will be placed in is digested with the same restriction endonuclease’s.
96
What is step 3 of molecular cloning after the cloning vector has been created?
The desired DNA fragment is ligated into the vector.
97
What is step 4 of molecular cloning after the desired DNA fragment is ligated into the vector?
The vector and DNA fragment complex are inserted into the host cell.
98
What is step 5 of molecular cloning after recombinant DNA molecule has been inserted into a host cell?
The recombinant DNA will replicate itself and this produces many copies (clones) in daughter cells.
99
What is step 6 of molecular cloning after the recombinant DNA has been cloned into daughter cells?
The recombinant DNA can be recovered from the colony of cells and it can then be analysed.
100
What are the 4 important features that each cloning vector must have?
Self replication.
A multiple cloning site.
Selectable markers.
Recovery of recombinant DNA.
101
How must cloning vectors be able to self repicate?
Before and after the insertion of foreign DNA.
102
What is the multiple cloning site that must be in a cloning vector?
It consists of a number of unique restriction sites that are located in the same area and nowhere else in the cell.
103
What are the selectable markers that must be in a cloning vector?
It will encode for a protein that will be selected for by the cell.
104
What is a good example of a selectable marker in a cloning vector?
A protein that codes for antibiotic resistance to a certain drug.
If the cell is grown in the presence of this drug then it has got to select the coning vector or it will die.
105
What organisms are plasmids usually used as cloning vectors for?
For bacteria.
106
What is the most common cloning vector?
Plasmids.
107
What is the major limitation of using a plasmid as a cloning vector?
They can only fit a limited amount of DNA inside them.
108
What are 2 features that plasmids must have if they are able to be used as a cloning vector?
Thye must contain an ORI and be able to replicate autonomously.
109
What is the function of a reporter gene in a recombinant plasmid?
It will signal whether a cell has taken up the plasmid.
110
How can we use an antibiotic to determine whether a bacterial cell has taken up a plasmid?
A plasmid contains antibiotic resistance and the bacterial cell is grown on a solution that contains the antibiotic.
If the cell has taken up the plasmid then it will be able to grow on the media.
111
What is a common gene that is used as a reporter gene in a recombinant plasmid?
The LAC-Z gene which helps bacteria to metabolise lactose.
112
Where will the restriction site be located if the LAC-Z gene is the reporter gene?
In the middle of the LAC-Z gene.
113
Why is the restriction site located in the middle of the LAC-Z gene if the LAC-Z gene is acting as a reporter gene?
The foreign DNA will bind to the restriction site and de-activate the LAC-Z gene.
114
How does the de-activation of the LAC-Z gene by the foreign DNA allow the LAC-Z gene to act as a reporter gene?
A plasmid that contains the gene of interest will not have a functional LAC-operon.
This means that the bacteria that take up the foreign DNA will form white colonies instead of blue colonies.
115
What kind of nutrient is used in the growth media if the LAC-Z gene is being used as a reporter gene?
X-GAL which contains a type of galactose that turns blue when cleaved.
This means that the bacteria with a functional LAC-Z gene will turn blue. Those that
116
Why will some plasmids not take up the foreign DNA after being cleaved with an endonuclease?
Because the restriction sites in the LAC-Z gene have sticky ends.
These ends can get back together before the foreign DNA is able to insert itself in the plasmid.
117
What are competent bacterial cells?
Altered bacterial cells that allow recombinant DNA to pass through the plasma membrane.
118
How are the cell walls of E.coli altered when tyring to make the cells competent?
Through the use of a salt such as calcium chloride or rubidium chloride.
This causes the cell wall to become more permeable.
119
When is the foreign DNA introduced to an E.coli cell that is becoming a comptent cell?
Once the plasmid has been mixed with the salt.
120
What step occurs after introducing foreign DNA to a recently salted ecoli cell that is becoming competent?
To perform heat shock, where the cell and foreign DNA are heated to around 40 degrees.
121
How does heat shock induce competency in an E.coli cell?
It allows the membrane to expand and the DNA can enter the cell.
The bacterial cells can then be spread on to nutrient agar and form a colony on the plate.
122
What are 2 other common forms of cloning vectors that can be used instead of plasmids?
Bacteriophage 𝛌.
Artificial chromosomes.
123
What is a mjor advantage of using bacteriophage 𝛌 as a vector instead of using a plasmid?
Bacteriophage 𝛌 can hold much larger piece’s of recombinant DNA than a plasmid.
124
What is commonly prepared through the use of bacteriophage 𝛌 as a cloning vector?
The preparation of genomic and cDNA libraries.
125
Why can bacteriophage 𝛌 hold a large recombinant gene?
Around 20 KB are removed the phage's dispensable region which creates space for the new DNA.
126
What is the dispensable region of a bacteriophage
A region of phage DNA that is not involved in the lytic cycle.
127
Cloning vectors that are inserted into the dispensable region of a phage are grown where?
These vectors can only be grown in the lab.
128
What will happen once the recombinant DNA has been inserted into bacteriphage 𝛌?
The bacteriophage injects the DNA into a bacterial cell and the bacterial cell will make the desired product.
129
What is the first step of using bacteriophage 𝛌 as a vector?
To remove the dispensable region and to form recombinant sequences from the DNA of interest.
130
How must the dispnesable region of a bacteriophage and the DNA containing the gene of interest be broken up?
The same restriction endonuclease must be used for both processes.
131
Which recombinant sequence is placed into bacteriophage 𝛌?
The recombinant sequence that contains the gene of interest.
132
How will a bacteriophage that contains recombinant DNA infect bacterial cells on an agar plate?
Via transduction.
133
What happens to the bacterial cells once they have been infected by a phage containing recombinant DNA?
The virus will replicate and eventually lyse the bacterial cell.
134
How can a colony that has been infected by a bacteriophage be visibly identified on an agar plate?
Infected colonies form clear plaques on an agar plate as there are no more cells growing there due to lysis.
135
What is found on the plaques created by bacteriophage 𝛌 in a cell culture?
Many baby viruses that contain the DNA of interest.
This means the amounts of the gene of interest has been amplified by viral replication inside the bacterial cell.
136
Why are artificial chromosomes used as cloning vectors?
As they can store extremely large sequences of DNA.
137
What purposes are artificial chromosomes used as cloning vectors for?
For mapping and analysing eukaryotic genomes such as in the human genome project.
For investigating genes that are located next to each other.
138
Where do the artificial chromosomes that are used as cloning vectors usually come from?
From yeast (YAC, yeast artificial chromosome).
From bacteria (BAC, bacterial artificial chromosome).
139
How much genetic data can artificial chromosomes hold?
Over 300 KB of inserted DNA.
140
What are BACs made from?
A bacterial plasmid.
141
What 2 features are always included on a BAC?
Recombinant DNA.
A fertility factor (F factor) which allows for lateral gene transfer.
142
What advantage does the F factor give to bacteria who have the plamsid?
The F factor allows bacteria to share the plasmid with other bacteria who do not have the plasmid.
143
How much foreign DNA can BACs hold?
Around 350 KB.
144
What shapes are YACs before they are digested by a restriction endonuclease?
Circular.
145
What shapes are YACs after they are digested by a restriction endonuclease?
Linear.
146
What is the name of the ORI in a YAC?
ARS (autonomously replicating sequence).
147
What 4 features do YACs contain?
An ARS (autonomously replicating sequence).
A centromere.
Telomeres.
Selectable markers located on each arm.
148
What are the selectable markers that are usually found on YACs?
URA-3 which encodes for the biosynthesis of uracil.
TRP-1 which encodes for the biosynthesis of tryptophan.
149
Where is the gene of interest inserted into a YAC?
On each side of the selectable markers.
150
How are reporter genes used in YACs?
The gene of interest is inserted into yeast cells that cannot form tryptophan or uracil.
The yeast cells are grown on a selective medium that does not contain tryptophan or uracil.
This means that only the cells that have taken up the YAC will be able to survive on this medium.
151
The choice of a cloning vector usually comes down to what?
How much DNA the scientist is looking to add to the vector.
152
The storage of DNA in vectors allows for what to be created?
DNA libraries.
153
How can an organisms entire genome be stored in a DNA library?
A collection of vectors can contain all of an organisms DNA.
154
How are cloning vectors stored in a DNA library?
In freezers and then the DNA of interest can be analysed at a later date.
155
How is each recombinant within a vector analysed?
Each recombinant can be copied and placed in an individual cell to be analysed.
156
What are the 2 kinds of DNA libraries?
DNA libraries that contain genomic DNA.
cDNA libraries that only contain genes that are expressed.
157
How will most genetic libraries store their genes of interest?
In such a way that the fragments will overlap and combine with each other.
158
Why are overlapping fragments stored in DNA libraries?
To allow scientists to form long sequences of DNA from the vectors in different plasmids.
This allows them to see how these sequences interact with each other.
159
What are the overlapping sequences that are used in gene libraries known as?
Partial digestions or contigs.
160
Has the DNA in partial digestions or contigs been broken up at every restriction site?
No, this creates the overlaps.
161
How is DNA sorted within a genomic library?
It is sorted into the order of the size of the DNA fragment.
162
How will DNA fragments move through an electrophoresis gel?
Small fragments will move quickly and will move futher through the gel than larger fragments.
163
Electrophoresis can sort what kind of DNA fragments into an order of size?
Electrophoresis can sort fragments between 100 BP and 50,000 BP into an order of size.
164
How is DNA normally viewed after electrophoresis has occurred?
Under UV light.
165
What is DNA stained with before it undergoes electrophoresis which allows it to show up under UV light?
Ethidium bromide.
166
What part of a gene is used to make a cDNA library?
The mRNA from the gene.
167
How is the mRNA from a gene examined?
When tissue that contains cells that express the gene is isolated from the body.
The mRNA is removed from the tissue and examined.
168
How is the mRNA removed from a tissue sample when creating a cDNA library?
By attaching a nucleotide linker that contains a bead of thymine residues to the poly-A tail.
The thymine and adenine residues form complimentary bonds and the mRNA can be removed.
169
What enzyme will create DNA from the mRNA of interest during the creation of a cDNA library?
Reverse transcriptase.
This allows us to see what strand of DNA codes for a particular RNA strand.
170
What kind of primer is used for the reverse transcriptase enzyme during the formation of a cDNA library?
A poly-T strand.
171
How many strands of DNA will reverse trascrpitase synthesise from a mRNA strand?
A single strand of DN which is always the coding strand (CDNA) and is covalently bound to the RNA strand.
172
What feature will reverse transcriptase form on the DNA that it synthesises?
A small fragment of DNA on the 3 prime end of the non-coding strand which is known as a Klenow fragment.
173
What use does the Klenow fragment have on a piece of DNA that has been synthesised by reverse transcriptase?
It serves as a primer for DNA polymerase.
174
What happens to the DNA/RNA complex after the DNA has been synthesised by a reverse transcriptase enzyme?
A digestion enzyme removes the RNA strand.
This allows DNA polymerase to bind to the Klenow fragment and synthesise the non-coding strand.
175
How is the Klenow fragment removed from a DNA molecule that has been synthesised from a piece of mRNA?
An S1-nuclease enzyme removes the Klenow fragment to form double stranded cDNA.
176
What molecules are used to help find a particular sequence of DNA in a DNA library?
A particular probe that is complimentary to the desired DNA fragment.
177
What is the process of searching for a particular fragment of DNA within a library known as?
Screening.
178
How can a probe indicate that is has matched up with a DNA fragment of interest?
It has a radioactive label or fluorescent marker that highlights the location of the desired fragment.
179
What are heterologous probes?
They are formed from a known gene and used to find an unknown gene.
E.g. if a gene from a dog is isolated, we can use that gene on a heterologous probe to see if the same gene is found in other organisms.
180
Do heterologous probes have to be completely complimentary?
They do not have to be completely complimentary, but they must have a high degree of complementarity.
181
What are homologous probes?
Probes that contain a sequence that is exactly complimentary to the sequence of interest.
182
What are degenerate probes?
Probes that contain a protein sequence that is used to identify a gene.
183
How is a degenerate probe assembled?
Edmans degradation removes each terminal amino acid from a polypeptide.
The triplet code for each amino acid is identified and the probes contain every combination of the possible triplet codes.
184
What are EST probes?
Probes that are made up of partial DNA sequences from cDNA libraries.
185
What are the partial sequences on an EST probe?
They contain the 5 prime or 3 prime end of the desired DNA and a small sequence of the protein coding region.
186
What are the 4 methods of labelling probes?
Random priming.
In vitro transcription.
Klenow fill in.
Oligonucleotides.
187
What is the random priming method of labelling a probe?
It involves adding radioactive nucleotides along the length of a fragment of DNA.
188
How are the radioactive nucleotides added to a random priming probe label?
The DNA is denatured to form single strands and a number of hexamers are added to one strand.
189
What are the names of the hexamers that are added to the single DNA strand by DNA polymerase I during the formation of a priming probe label?
Radioactive dNTPs known as DATP, DTTP, DGTP, DCTP.
190
How does in vitro transcription act as a probe label?
A sequence of radioactive nucleotides are complimentary to the desired DNA strand.
191
How is the RNA labelled during the formation of an in vitro transcription probe?
The RNA is labelled while it is being transcribed from a cDNA template.
192
How is an in vitro transcription probe label synthesised?
By the host cell machinery.
193
What is a Klenow fill in probe label?
A restriction endonuclease digests a short segment on the 3-prime ends of both DNA strands creating 5 prime overhangs.
The Klenow fragment of DNA polymerase I fills in the short sequences at the 3 prime end using radioactive nucleotides.
194
How are oligonucleotide probe labels formed?
By adding an ATP molecule that has a radioactive gamma phosphate to the 5 prime end of a probe.
This is done via the use of the enzyme T4 polynucleotide kinase.
195
How is DNA library screening carried out?
The bacteria that contain the gene library are plated.
Some of the bacterial colonies are transferred to a membrane.
These colonies undergo cracking which exposes de-natured DNA.
The denatured DNA can be hybridised with a radioactive probe.
If the probe hybridise's with the DNA then double stranded DNA molecules will be visible after X ray.
The radioactive strand makes up one strand and the original strand makes up the other.
