recombinant DNA technology- chapter 21 Flashcards
1
Q
what do recombinant DNA technology do
A
- allows genes to be manipulated, altered and transferred from organism to organism.
- These techniques have enabled us to understand better how organisms work and to design new industrial processes and medical applications.
- A number of human diseases result from individuals being unable to produce for themselves various metabolic chemicals
2
Q
explain recombinant DNA
A
The DNA of two different organisms that have been combined by isolating genes, cloning them and transferring them to microorganisms
3
Q
what is the resulting organism known as
A
transgenic or genetically modified organism (GMO)
4
Q
what are the 5 steps of the processes of the recombinant DNA technology
A
- Isolation of the DNA fragments that have the gene for the desired protein.
- Insertion of the DNA fragment into a vector.
- Transformation, transfer of DNA into suitable host cells.
- Identification of the host cells that have successfully taken up the gene by use of gene markers.
- Growth/cloning of the population of host cells
5
Q
what are 3 methods of producing DNA fragments
A
- Conversion of mRNA to cDNA using reverse transcriptase
- Using restriction endonucleases to cut fragments containing the desired gene from DNA.
- Creating the gene in a gene machine, usually based on a known protein structure
6
Q
how does using reverse transcriptase happen (short)
A
Catalyses the production of DNA from RNA (this is the reverse of a normal transcriptase)
7
Q
process of reverse transcriptase to produce DNA fragments
A
- cell that readily produces the protein is selected (e.g. B-cells of the islets of Langerhans)
- These cells have large quantities of the relevant mRNA, which is therefore more easily extracted.
- Reverse transcriptase is then used to make DNA from RNA. This is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA.
- To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene
8
Q
process of using restriction endonucleases to produce DNA fragments
A
- Some bacteria defend themselves against viruses by producing enzymes that cut up the viral DNA- these are restriction endonucleases
- There are many types, each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence.
- Sometimes, this cut occurs between two opposite base pairs, leaving two straight edges known as blunt ends.
- Other restriction endonucleases cut DNA in a staggered fashion, which leaves an uneven cut so each strand of the DNA has exposed unpaired bases.
9
Q
explain the process of the ‘gene machine’
A
- Desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce. The amino acid sequence of the protein is determined. From this, the mRNA codons are looked up and complementary DNA triplets are worked out.
- The desired sequence of nucleotide bases for the gene is fed into a computer.
- The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements.
- The computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene.
- In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence.
- The oligonucleotides are then joined together to make a gene. This gene doesn’t have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction.
- The PCR also constructs the complementary strand of nucleotides to make the required double stranded gene. It then multiples his gene many times to give numerous copies.
- Using sticky ends the gene can then be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organisms in the future.
- The genes are checked using standard sequencing techniques and those with errors are rejected.
10
Q
what are 2 advantages of the ‘gene machine’
A
- Can be produced in a short amount of time (as little as 10 days) with great accuracy.
- A further advantage is that these artificial genes are also free of introns and other non-coding DNA so can be transcribed and translated by prokaryotic cells
11
Q
what is the polymerase chain reaction (PCR)
A
- A method of copying fragments of DNA.
- Process is automated, rapid and efficient
12
Q
what does the process of polymerase chain reaction require (5)
A
- DNA fragment to be copied
- DNA Polymerase- this joins together tens of thousands of nucleotides in a matter of minutes. Taq polymerase is obtained from bacteria in hot springs so it is able to tolerate heat and does not denature.
- Primers- short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.
- Nucleotides- which contains each of the four bases found in DNA.
- Thermocycler- a computer-controlled machine that varies temperatures precisely over a period of time.
13
Q
how is the polymerase chain reaction carried out (3 stages)
A
- Separation of the DNA strand- DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. Temperature is increased to 95C, causing the two strands of the DNA fragments to separate due to the breaking of the hydrogen bonds.
- Addition (annealing) of the primers- mixture is cooled to 55C, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment. 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. Primers also prevent the two separate strands from rejoining.
- Synthesis of DNA – The temperature is increased to 72C. This is optimum temperature 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 sequence until it reaches the end of the chain.
14
Q
2 advantages of in vitro gene cloning
A
- Extremely rapid- 100 billion copies within a few hours. Only a minute amount of DNA is needed as PCR can quickly increase the amount, although any contaminating DNA found will also be increased. In vivo would take many days or weeks to produce the same quantity.
- Does not require living cells- all that is required is a base sequence of DNA.
15
Q
how do you find the fragment that has the required gene and explain
A
- using a DNA probe
- Once the fragment with the gene has been obtained, the next stage is to clone it so that there is a sufficient quantity for medical or commercial use.
- This can be achieved by:
1. In vivo, by transferring the fragments to a host cell using a vector.
2. In vitro, using the polymerase chain reaction
16
Q
what are recognition sites
A
The sequence of DNA that are cut by restriction endonucleases
17
Q
what happens if the sequence of DNA is cut in a staggered fashion
A
- the cut ends of the DNA double strand are left with a single strand which is a few nucleotide bases long
18
Q
what happens if the same restriction endonuclease is used to cut the DNA
A
- then all the fragments produced will have ends that are complementary to one another
- This means that the single-stranded end of any one fragment can be joined (Stuck) to the single-stranded end of any other fragment (their ends are sticky)
- Once the complementary bases of two sticky ends have paired up, an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the two sections of DNA
19
Q
why are sticky ends important
A
because if the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism
20
Q
what does the preparation of DNA fragments for insertion
A
the addition of extra lengths of DNA
21
Q
what is needed for the transcription of any gene to take place
A
the enzyme that synthesises mRNA (RNA polymerase) must attach to the DNA near a gene
22
Q
what is a promoter
A
The binding site for RNA polymerase is a region of DNA
23
Q
what is a terminator
A
- Another region release RNA polymerase and ends transcription
- need to add a terminator to the other end of our DNA fragment to stop transcription and appropriate point
24
Q
how to insert the DNA fragments into a vector
A
- We now need to insert the DNA fragment with promoter and terminator regions into a carrying unit known as a vector.
- This is used to transport the DNA into the host cell.
- There are different types of vectors but the most commonly used is the plasmid, which are circular lengths of DNA which are separate from the main bacterial DNA.
- Plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic resistance genes to break the loop.
- The restriction endonuclease used is the same as the one that cut out the DNA fragment, ensuring the sticky ends are complementary.
- When the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them, where they are incorporated, the join is made permanent using the enzyme DNA ligase.
25
what is transformation
- They are plasmids which must be reintroduced into bacterial cells, this is called transformation, and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.
- The calcium ions and changes in temperature make the bacterial membrane permeable allowing the plasmids to pass through into the cytoplasm.
26
why are the 3 reasons for not all bacteria cells possessing the DNA fragments with the desired gene for the desired protein
1. Only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together.
2. Some plasmids will have closed up again without incorporating the DNA fragment.
3. Sometimes the DNA fragment ends join together to form its own plasmid.
27
explain the process for using the gene for antibiotic resistance to identify which bacterial cells have taken up the plasmid
what is the disadvantage of this
1. All the bacterial cells are grown on a medium that contains the antibiotic ampicillin.
2. Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance.
3. These bacteria cells are able to break down the ampicillin and therefore survive.
4. The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die.
some of them will have closed up without incorporating the new gene- these will also survive
28
explain marker genes
- All methods involve using a second, separate gene on the plasmid. This second gene is easily identifiable for one reason or another.
E.g.
1. It may be resistant to an antibiotic.
2. It may make a fluorescent protein that is easily seen.
3. It may produce an enzyme whose action can be identified.
29
what is the problems with antibiotic resistance marker genes
- outdated method
- it destroys the very cells that contain the required gene
30
what is replica planting
- to identify the cell
- This uses the other antibiotic-resistance gene in the plasmid, the gene that was cut in order to incorporate the required gene
31
explain fluorescent markers
- More rapid method is the transfer of a gene from jellyfish into the plasmid. It produces a green fluorescent protein (GFP).
- The gene to be cloned is transplanted into the centre of the GFP gene.
- Any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP.
- Bacterial cells that have not taken up the gene will continue to produce GFP and to fluoresce
- Cells are viewed under a microscope and the cells that do not fluoresce are retained.
32
explain enzyme markers
- Produce the enzyme lactase.
- This will turn a particular substrate blue.
- The required gene is transplanted into the gene that makes lactase.
- If the plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase so if grown on a colourless substrate they will be unable to change its colour.
33
what are DNA probes
Is a short, single stranded length of DNA that has some sort of label attached that makes it easily identifiable
34
what are 2 most commonly used DNA probes
1. Radioactively labelled probes- which are made up of nucleotides with the isotope 32-P. The probe is identified using x-ray film that is exposed by radioactivity.
2. Fluorescently labelled probes- which emit light (fluoresce) under certain conditions, for instance when the probe has bound to the target DNA sequence.
35
how do DNA probes identify alleles of genes
1. A DNA probe is made that has base sequences that are complementary to part of the base sequence of the DNA that makes up the allele of the gene that we want to find.
2. The double-stranded DNA that is being tested is treated to separate its two strands.
3. The separated DNA strands are mixed with the probe, which binds to the complementary base sequence on one of the strands. DNA Hybridisation.
3. The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.
36
what is DNA hybridisation
- Takes place when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.
- Before this happens, the two strands of DNA must be separated- By heating (denaturation) until the strands separate. When cooled, the complementary bases on each strand recombine (anneal) with each other to reform the original double strand. Given time, all strands in the mixture of DNA will pair up with their partners.
37
how do you locate a specific allele of a gene by using DNA probes and DNA hybridisation
1. Determine the sequence of nucleotide bases of the mutant allele we are trying to locate- achieved using DNA sequencing techniques.
2. A complementary fragment of DNA is produced.
3. Multiple copies of our DNA probe are formed using PCR.
4. A DNA probe is made by attaching a marker to the DNA fragment.
5. DNA from the person suspected of having the mutant allele we want to locate is heated to separate its two strands.
6. The separated strands are cooled in a mixture containing many of our DNA probes.
7. If the DNA contains the mutant allele, one of our probes is likely to bind to it.
8. The DNA is washed clean of any unattached probes.
9. The remaining hybridised DNA will not be fluorescently labelled with the dye attached to the probe.
10. The dye is detected by shining light onto the fragments causing the dye to fluoresce which can be seen with a microscope.
38
why is genetic screening important
- It is important to screen individuals who may carry a mutant allele.
- Such individuals usually have family history of a disease.
- Screening can determine the probabilities of a couple having offspring with a genetic disorder.
- Potential parents who are at risk can obtain advice from a genetic counsellor about the implications of having children, based on their family history and the results of genetic screening.
- It also valuable in the detection of oncogenes.
- Some peoples can inherit mutated tumour suppressor genes and so these individuals are at greater risk of developing cancer.
- If a mutated gene is detected by genetic screening, individuals who are at greater risk of cancer can then make informed decision about their lifestyle and future treatment.
- Genetic screening allows doctors to provide advice and health care based on an individuals genotype.
- Some peoples genes can mean that a particular drug may be either more or less effective in treating a condition.
- Doctors and pharmacists can determine, more ,exactly, the dose of a drug which will probe the desired outcome.
39
what is the process of genetic screening
1. It is possible to fix hundreds of different DNA probes in an array (pattern) on a glass slide.
2. By adding a sample of DNA, any complementary DNA sequence in the donor DNA will bind to one or more probes.
3. This makes is possible to test simultaneously for many different genetic disorders by detecting fluorescents that occurs where binding has taken place.
40
41
what is genetic counselling and why its used
- Advice and information are given that enable people to make personal decisions about themselves or their offspring.
- One aspect is to research the family history of an inherited disease and to advise parents on the likelihood of it arising in their children.
- A counsellor can inform the couple of the effects a disease they may carry as well as its emotional, psychological, medical, social and economic consequences.
- On this advice, the couple can then choose whether or not to have children.
- Counselling can also make them aware of any further medical tests that might give a more accurate prediction of whether their children will have the condition e.g IVF and screening.
42
what is genetic fingerprinting
- Diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis.
- The technique relies on the fact that the genome of most eukaryotic organisms contains may repetitive, non-coding bases of DNA.
- DNA bases that are non-coding are known as variable number tandem repeats (VNTRs).
- For every individuals the number of length of VNTRs has a unique pattern.
- The more closely related two individuals are, the more similar the VNTRs will be.
43
what is electrophoresis and explain how it works
- Is used to separate DNA fragments according to their size.
- The DNA fragments are placed on to an agar gel and a voltage is applied across it.
- The resistance of the gel means that the larger the fragments, the more slowly they move.
- DNA fragments of different lengths are separated.
- If the DNA fragments are labelled, their position in the gel can be determined by placing a sheet of X-ray film over the agar gel for several hours.
- The radioactivity from each DNA fragment exposes the film and show where the fragment is situated on the gel.
- Only DNA fragments up to around 500 bases long can be sequenced this way.
- Larger genes and whole genomes need to be cut into smaller fragments by restriction endonucleases.
44
what are the stages of genetic fingerprinting (5) and explain
1. Extraction- sample of animal tissue (can be a tiny amount), the DNA is extracted by separating it from the rest of the cell. The quantity of DNA can be increased by the PCR.
2. Digestion – DNA is then cut into fragment, using the same restriction endonucleases. They are chosen to cut close to the target DNA.
3. Separation – the fragments of DNA are next separated according to size by gel electrophoresis under the influence of electrical voltage. Gel is then immersed in alkali in order to separate the double strands into single strands.
4. Hybridisation – DNA probes are not used to bind with VNTRs. The probes and VNTRs are complementary and bind under specific conditions such as temperature or pH. Process is carried out with different probes.
5. Development – an x-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes. Because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bands are revealed.
45
how are the results from genetic fingerprinting interpreted
- DNA fingerprints from two samples are visually checked.
- If there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculate the length of the DNA fragments from the bands.
It does this using data obtained by measuring the distances travelled during electrophoresis by known lengths of DNA.
- Finally, the odds are calculated of someone else having an identical fingerprint. The closer the match between the two patients, the greater the probability that the two sets of DNA have come from the same person.
46
how is DNA fingerprinting used to see genetic relationship and variability
- Can be used to help resolve questions of paternity.
- Individuals inherit half the genetic material from their mother and half from their father.
- Therefore each band on a DNA fingerprint of an individual should have a corresponding band in one of the parents DNA fingerprint.
- Also useful in determining genetic variability within a population, the more closely two individuals are related the closer the resemblance of their genetic fingerprints.
- A population whose members have very similar genetic fingerprints has little genetic diversity. Greater variety of genetic fingerprints has greater genetic diversity.
47
how is DNA fingerprinting used for forensic science
- DNA is often left at the scene of a crime.
- Genetic fingerprinting can establish whether a person is likely to have been present at the crime scene, although this does not prove they actually carried out the crime.
- The probability that someone else’s DNA might match that of the suspect has to be calculated. This is based on the assumption that the DNA which produces the banding patterns is randomly distributed in the community.
48
how is DNA fingerprinting used for medical diagnosis
- Can help in diagnosing diseases such as Huntington’s disease- disease of the nervous system that results from a three-base sequence (AGC) at one end of a gene on chromosome 4 being repeated over and over again.
- A sample of DNA from a person with the allele for Huntington’s diseases can be cut with restriction endonucleases and a DNA fingerprint prepared.
- This can then be matched with fingerprints of people with various forms of the disease and those without the disease.
- In this way, the probability of developing the symptoms, and when, can be determined.
- Also used to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with that of known pathogens.
49
how is DNA fingerprinting used for plant and animal breeding
- Can be used to prevent undesirable inbreeding during breeding programmes on farms or in zoos.
- It can also identify plants or animals that have a particular allele of a desirable gene.
- Individuals with this allele can be selected for breeding in order to increase the probability of their offspring having the characteristic that it produces.
- Another application is the determination of paternity in animals and thus establishing the pedigree (family tree) of an individual.
