Recombinant DNA technology 2

?
  • Created by: Hindleyc
  • Created on: 20-04-19 15:27
Having cut the DNA into fragments what is it necessary to find
the fragment which has the required gene amongst all the rest
1 of 105
how is this done
using a DNA probe
2 of 105
Once fragment with the gene has been obtained what's the next stage
to clone it so that there is sufficient quantity for medical or commercial use
3 of 105
how can this be achieved
in vivo- by transferring the fragment to a host cell using a vector or in vitro using the polymerase chain reaction
4 of 105
Before we can consider how genes can be cloned within living organisms (in vivo cloning) what is important
the sticky ends left when RNA is cut by RE
5 of 105
What are the sequences of DNA that are cut by RE called
recognition sites
6 of 105
If the Recognition site is cut in a staggered fashion the cut ends of the DNA double strand are left with
a single strand which is a few nucleotide bases long
7 of 105
What are the nucleotides on the singles strand at one side of the cut
complementary to those at the other side because they were previously paired together
8 of 105
If the same RE is used to cut DNA then all the fragments produced will
have ends that are complementary to one another
9 of 105
What does this mean
that single stranded end of any one fragment can be joined (stuck) to the single-stranded end of any other fragment- in other words their ends are sticky
10 of 105
Once the complementary base of 2 sticky ends have paired up what enzyme is used to bind the phosphate-sugar framework of the 2 sections of DNA and so unite them as 1
DNA ligase
11 of 105
What do sticky ends have and why
considerable importance, provided the same RE is used we can combine the DNA of 1 organism with that of any other organism
12 of 105
What does the preparation of the DNA fragment involve
the addition of extra lengths of DNA
13 of 105
For transcription of any gene to take place, what must happen
the enzyme that synthesises mRNA (RNA polymerase) must attach to the DNA near a gene
14 of 105
What is the binding site or RNA polymerase
region of DNA known as a promoter
15 of 105
What do nucleotide bases of the promotor attach
both RNA polymerase and transcription factors and so begin the process of transcription
16 of 105
If we want our DNA fragment to transcribe mRNA in order to make a protein, what is essential
that we attach to it the necessary promotor region to start the process
17 of 105
In the same way as a region of DNA binds RNA polymerase and begins transcription of a gene, what do another region of DNA do
releases RNA polymerase and ends transcription
18 of 105
What is this called and what do we need to do
terminator and we need to add a terminator region to the other end of our DNA fragment to stop transcription at the appropriate point
19 of 105
Once an appropriate DNA fragment has been cut from the rest of the DNA and the promotor and terminator regions added what is the next task
join it into a carrying unit, known as a vector
20 of 105
What is this vector used to
transport the DNA into the host cell
21 of 105
What are there different types of
vector but most commonly used is the plasmid
22 of 105
what's a plasmid and what does it contain
circular lengths of DNA found in bacteria which are separate from the main bacterial DNA and almost always contain genes for AB resistance and RE are used at one of these AB resistance genes to break up the plasmid loop
23 of 105
What RE is used
the same as the one that cut out the DNA fragment this ensures that the sticky ends of the opened-up plasmid are complementary to the sticky ends of DNA fragment
24 of 105
When the DNA fragments are mixed with opened up plasmids what may happen
they may become incorporated into them
25 of 105
where they are incorporated how is the join made permanent
using the enzyme DNA ligase
26 of 105
What do these plasmids now have
recombinant DNA :)
27 of 105
Once the DNA has been incorporated into at least some of the plasmids and must they then be
reintroduced into bacterial cells
28 of 105
What is this process called
transformation
29 of 105
what does it involve
the plasmids and bacterial cells being mixed together in a medium containing calcium ions
30 of 105
What do the calcium ions and changes in temperature make the bacterial membrane
permeable allowing the plasmids to pass through the CSM into the cytoplasm
31 of 105
However why will not all the bacterial cells possess the DNA fragments with the desired gene for the desired protein
only a few bacterial cells (As few as 1%) take up the plasmids when the 2 are mixed together, some plasmids will have closed up again without incorporating the DNA fragment and sometimes the DNA fragment ends join together to form its own plasmid
32 of 105
What is the first task
to identify which bacterial cells have taken up the plasmids
33 of 105
what is one to do this
to use the fact that bacteria have evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the AB before it can destroy the bacterium-
34 of 105
where are genes for the production of these enzymes are found
in the plasmids
35 of 105
What do some plasmids carry genes for
resistance to more than one AB
36 of 105
What does the task of finding out which bacterial cells have taken up the plasmid entail
using the gene for AB resistance which is unaffected by the introduction of the new gene
37 of 105
How does the process work
all bacterial cells are grown on a medium that contains AB ampicillin, bacterial cells that have taken up the plasmids will have acquired gene for A resistance
38 of 105
What will these bacterial cells be able to
break down the A and therefore survive
39 of 105
The bacterial cells that haven't taken up the plasmids will not be
resistant to A and therefore die
40 of 105
What is this an effective method of showin
which of the bacterial cells have taken up the plasmids
41 of 105
However some cell will have
taken up the plasmid which closed up without incorporating the new gene and these cells will have also survived
42 of 105
what is next task
to identify the cell without the new gene and eliminate them
43 of 105
how is this achieved
using marker genes
44 of 105
What are there
a number of different ways of using marker genes to identify whether a gene has been taken up by bacterial cells
45 of 105
What do they all involve using
a second , separate gene on the plasmid
46 of 105
what is this second gene
easily identifiable for one reason or another
47 of 105
such as
may be resistant to an antibiotic, may make a fluorescent protein that is easily seen, may produce an enzyme whose action can be identified
48 of 105
What has the use of AB resistance marker genes as markers
rather old technology and has been superseded by other methods
49 of 105
However what is it
an interesting example of how science works- particularly of the way in which scientists use knowledge and understanding to solve new problems and use appropriate methodology and carry out relevant experiments
50 of 105
To identify those cells with plasmids that have taken up the new gene what do we use
a technique called replica plating
51 of 105
What does this process use
the other AB resistant gene in the plasmid- the gene was cut in order to incorporate the required gene eg gene for resistance to Tetracyline
52 of 105
As this gene has been cut
it will no longer produce the enzyme that breaks down tetracycline (in other words bacteria that have taken up the req gene will no longer be resistant to T so we can therefore identify these bacteria by growing them on a culture that contains T
53 of 105
Problem ?
Treatment with T will destroy the very cells that contain the required gene
54 of 105
However using what technique is it possible to identify living colonies of bacteria containing the required gene
Replica plating :)
55 of 105
What is another gene marker
The gene that produces the enzyme lactase
56 of 105
What will lactase turn
a particular colourless substrate blue
57 of 105
Again what is the required gene
transplanted into the gene that makes lactase
58 of 105
So if a plasmid with the required gene is present in bacterial cell the colonies grown from it
will not produce lactase therefore when these bacterial cells are grown on the colourless substrate they will be unable to change its colour
59 of 105
Where the gene has not been taken up by the bacteria
they will turn the substrate blue and these bacteria can be discounted
60 of 105
What is a more recent and more rapid method is
the transfer of a gene from a gene from a jellyfish into the plasmid
61 of 105
what does the gene in question do
produce green fluorescent protein (GFP)
62 of 105
Gene to be cloned is?
transported into the centre of the GFP gene so any bacterial cells that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP
63 of 105
Bacterial cells that have not taken up the gene will
continue to produce GFP and to fluoresce
64 of 105
Unlike the cell that the cells that have not taken up the gene these cells have taken it up will
not fluoresce
65 of 105
As the bacterial cells with the desired gene are not killed there
is no need for replica plating
66 of 105
Results can be obtained by simply
viewing the cells under a microscope and retaining those that don't fluoresce
67 of 105
What does this make the process
more rapid
68 of 105
What in Vitro gene cloning
the polymerase chain reaction
69 of 105
What is PCR
Method of copying fragments of DNA
70 of 105
What is the process
Automated making it both rapid and efficient
71 of 105
What does the process require
The DNA fragment to be copied , DNA polymerase (enzyme capable of joining together 10s of 1000s of nucleotides in a matter of minutes- one such enzyme taq polymerase is obtained from bacteria in hot springs
72 of 105
why taq
therefore tolerant to heat (thermostable) and doesn't denature during the high temperatures used as part of the process
73 of 105
what else needed
Primers (short sequences of nucleotides that have a set of bases complementary to those at one end of each of the 2 DNA fragment) Nucleotides which contain each of the 4 bases found in DNA and Thermocyler
74 of 105
What is a thermocycler
a computer controlled machine that varies temperature precisely over a period of time
75 of 105
what are the 3 stages
Separation of the DNA strand, Addition (annealing) of the primers, Synthesis of DNA
76 of 105
Step 1
DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler temp inc to 95*c causing the 2 strands of DNA fragments to separate due to the breaking of the H bonds between the 2 DNA strands
77 of 105
step 2
Mixture is cooled to 55*c causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment
78 of 105
What do the primers provide
the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an EXISTING chain
79 of 105
What can primers also do
prevent the 2 separate strands from simply rejoining
80 of 105
step 3
Temp is increased to 72* This is the optimum temp for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands- it begins at the primer on both strands and adds the nucleotides in a sequence
81 of 105
until
until it reaches the end of the chain
82 of 105
Because both strands are copied simultaneously there are now
2 copies of the original fragment
83 of 105
once the 2 DNA strands are completed
the process is repeated by subjecting them to the temperature cycle again resulting in 4 strands
84 of 105
How long does the whole temperature cycle take
around 2 minutes
85 of 105
over how many copies of the DNA can be made in only 25 temp cycles
1 million
86 of 105
How many in just a few hours
100 billion copies
87 of 105
The PCR has
revolutionised many aspects of science and medicine
88 of 105
why
even tiniest sample of DNA from a single hair or a speck of blood can now be multiplied to allow forensic examination and accurate cross matching
89 of 105
Advantages of in vitro gene cloning
Extremely rapid- within a matter of hrs 100 billion copies of a gene can be made which is particularly valuable where only a minute amount of DNA is available eg at the scene of a crime
90 of 105
what can this quickly be
increased using the PCR and so there is no loss of valuable time before forensic analysis and matching can take place
91 of 105
what's a complicating factor
PCR will also increase massively any other contaminating DNA found at the scene
92 of 105
What would in vivo cloning take
many days or weeks to produce the same quantity of DNA
93 of 105
Another ADV
Doesn't require living cells- all that is required is a base sequence of DNA that needs amplification and no complex culturing techniques requiring time and effort are needed
94 of 105
Advantages of in vivo gene cloning are
Particularly useful where we wish to introduce a gene into another organism as it involves the use of vectors once we have introduced the gene into a plasmid this plasmid can be used to deliver the gene into another organism, such as human being
95 of 105
eg
it can transform other organisms- this is done using a technique called gene therapy
96 of 105
What does it involve (adv)
Almost no risk of contamination- gene that has been cut by the same RE can match the sticky ends of the opened up plasmid. contaminant DNA will therefore not be taken up by the plasmid
97 of 105
what do in vitro cloning require
a v pure sample bc any contaminant DNA will also be multiplied and could lead to a false result
98 of 105
other ADV
v accurate bc DNA copied has few if any errors- at one time about 20% of the DNA cloned in vitro by the PCR was copied innacurateyl but modern techniques have improved the accuracy of the process considerably
99 of 105
However
any errors in copying DNAor any contaminants in the sample will also be copied in subsequent cycles- this problem hardly ever arises with in vivo cloning because although mutations can arise these are very rare
100 of 105
Another ADV
cuts out specific genes so it is therefore a v precise procedure as the culturing of transformed bacteria provides many copies of a specific gene and not just copies of the whole DNA sample
101 of 105
What does in vivo also do
produce transformed bacteria that can be used to produce a large quantities of gene products- transformed bacteria can produce proteins for commercial or medical use eg hormones such as insulin
102 of 105
Evaluate gene tech adv
Microorganisms can be modified to produce a range of substances, microorganisms can be used to control pollution, GM plants can be transformed to produce a specific substance, GM crops can be engineered to have financial and environmental advantages,
103 of 105
AND
GM crops can help prevent certain diseases, GM animals are able to produce expensive drugs, ABs, hormones and enzymes relatively cheaply . Replacing defective genes (gene therapy) might be used2cure certain g disorders AND GF can be used in forensicS
104 of 105
risks of recombinant DNA technology
impossible to predict with complete accuracy what the ecological consequences will be, Recombinant gene may pass from the organism it was placed in to a completely different one, Any manipulation
105 of 105

Other cards in this set

Card 2

Front

how is this done

Back

using a DNA probe

Card 3

Front

Once fragment with the gene has been obtained what's the next stage

Back

Preview of the front of card 3

Card 4

Front

how can this be achieved

Back

Preview of the front of card 4

Card 5

Front

Before we can consider how genes can be cloned within living organisms (in vivo cloning) what is important

Back

Preview of the front of card 5
View more cards

Comments

No comments have yet been made

Similar Biology resources:

See all Biology resources »See all DNA, genetics and evolution resources »