Producing DNA Fragments
Recombinant DNA technology: the processes by which genes are manipulated, altered or transferred from organism to organism.
The process of making protein using the DNA technology of gene transfer and cloning involves a number of stages:
- isolation of the DNA fragments that have the gene for the desired protein
- insertion of the DNA fragment into a vector
- transformation: the transfer of DNA into suitable host cells
- identification of the host cells that have successfully taken up the gene by the use of gene markers
- growth/cloning of the population of host cells
Before a gene can be transplanted it muct be identified and isolated from the rest of the DNA. Two of the methods employed use enzymes: reverse transcriptase and restriction endonucleases
Using Reverse Transcriptase
Reverse transcriptase catalyses the production of DNA from RNA.
- a cell that readily produces the protein is selected
- these cells have large quantities of the relevant mRNA, which is extracted
- reverse transcriptase is used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because is it made up of the nucleotides that are complementary to the mRNA
- to make the other strand of DNA, DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene.
Using Restriction Endonucleases
Some bacteria defend themselves from viruses that inject their DNA into them by producing enzymes. These enzymes cut up the viral DNA and are restriction endonucleases.
Each restriction endonuclease 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, leaving an uneven cut where each strand of DNA has exposed, unpaired bases. The two sequences are opposites of one another and so are a palindrome. These are called sticky ends.
In Vivo Gene Cloning
Importance of 'sticky ends'
Once the fragments of DNA have been obtained, the next stage is to clone them so that there is a sufficient quantity for medical use. This can be achieved in two ways: in vivo, by transferring the fragments to a host cell using a vector or in vitro, by using the polymerase chain reaction.
The sequences of DNA that are cut by restriction endonucleases are called recognition sites. If the recognition site is cut in a staggered fashion, the cut end of the DNA double strand are left with a single strand which is a few nucleotide bases long.
If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complementary to one another. So the single-stranded end of any one fragment can be joined to the single-stranded end of any other fragment. Once the complementary bases of two sticky ends have paired up, DNA ligase is used to join the phosphate sugar framework of the two sections of DNA.
We can combine the DNA of one organism with that of any other organism.
In Vivo Gene Cloning
Insertion of DNA fragment into a vector
Once an appropriate fragment of DNA has been cut, it needs to be joined to a vector. A vector, for example a plasmid, is used to transport DNA into the host cells. Plasmids normally contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic resistance genes to break the plasmid loop. The restriction endonuclease used in the same as the one that cut out the DNA fragment, therefore the ends of the plasmid are complementary to the ends of the DNA fragment. So when the DNA fragments are mixed with the plasmids, they become incorporated into them, which is made permanent by DNA ligase. These plasmids now have recombinant DNA.
In Vivo Gene Cloning
Introduction of DNA into host cells
Once the DNA has been incorporated into some plasmids, they must then be reintroduced into bacterial cells. This is 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 bacteria permeable, allowing plasmids to pass through the cell membrane into the cytoplasm. However, not all the bacterial cells will possess the DNA fragments because:
- only a few bacterial cells take up the plasmids when mixed together
- some plasmids will close up before incorporating the DNA fragment
Some plasmids carry genes for resistance to two antibiotics: ampicillin and tetracycline.
- all the bacterial cells are grown on a medium that contains the antibiotic ampicillin
- bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance and so are able to break down the ampicillin and survive
- the bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and so die
In Vivo Gene Cloning
Some cells will have taken up the plasmid and closed up without encorporating the new gene and these will have also survived. To identify these cells and eliminate them we use gene markers.
Gene markers use a second, seperate gene on the plasmid. This gene is easily identifiable because:
- it may be resistance to an antibiotic
- it may make a flourescent protein that is easily seen
- it may produce an enzyme whose action can be identified
In Vivo Gene Cloning
To identify those cells with plasmids that have been taken up the new genes we use replica plating. This uses the antibiotic resistance gene that was cut. For example, the gene for resistance to tetracycline was cut and so the gene will no longer produce the enzyme that breaks tetracycline down so it will no longer be resistant. So we identify these bacteria by growing them on a culture that contains tetracycline. But as tetracycline will identify but destroy the cells that contain the required gene, replica plating is used:
- the bacteria cells that survive treatment with ampicillin have taken up the plasmid
- these cells are cultured by spreading them thinly on agar plates
- each seperate cell on the plate will grow into a genetically identical colony
- a tiny sample of each colony is transferred onto a second plate in the same position as the origional
- this replica plate contains the second antibiotic
- the colonies killed by this antibiotic must have taken up the required gene
- the colonies in exactly the same position on the origional plate are the ones that possess the required gene. These therefore are bacteria that have been genetically modified: transformed.
In Vivo Gene Cloning
The transference of a gene from jellyfish that produces a green flourescent protein can also be used. The gene to be cloned is transplanted into the centre of the flourescent gene. So any bacterial cell that has taken up the plasmid with the gene to be cloned will not flouresce.
As the bacterial cells with the desired gene are not killed, there is no need for replica plating.
Another gene marker is the gene that produces the enzyme lactase. Lactase will turn a colourless substrate blue. So the required gene is transplanted into the gene that makes lactase. If a plasmid with the required gene is present in a bacterial cell, then the colonies will not produce lactase. So when these bacterial cells are grown on the colourless substrate, they will not change its colour. When the gene has not transformed the bacteria, the colonies will turn the substrate blue.
In Vitro Gene Cloning
Polymerase Chain Reaction is a method of copying fragments of DNA. It requires:
- the DNA fragment to be copied
- DNA polymerase to join together the nucleotides
- primers: sequences of nucleotides that are complementary ends of each of the fragments
- thermocycler: a computer controlled machine that varies temperatures precisely over a period of time
PCR has three stages:
- Separation of DNA strand: DNA fragments, primers and DNA polymerase are heated at 95 degrees inthe thermocycler, causing the DNA fragments to separate
- Addition (annealing) of the primers: the mixture is cooled to 55 degrees, causing primers to join to their complementary bases at the ends of the fragment. They provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attatch nucleotides to the end of an existing chain
- Synthesis of DNA: temperature is increased to 72 degrees for DNA polymerase to add complementary nucleotides along each strand, beginning at the primers
- extremely rapid (helpful in some areas, e.g crimes)
- does not require living cells
- useful for introducing a gene into another organism as it uses vectors (e.g gene therapy)
- no risk of contamination as the same restriction endonuclease can match the plasmid with the gene so no contaminant DNA will be taken up
- very accurate as it is very rare that the DNA copied has errors. PCR can often have errors, which are copied in subsequent cycles
- cuts out specific genes
- produces transformed bacteria that can be used to produce large quantities of gene products e.g the hormone insulin
Genetic modifications can benefit humans in many ways:
- increasing the yield from animals or plant crops
- improving the nutrient content of foods
- introducing resistance to disease and pests
- making crop plants tolerant to herbicides
- developing tolerance to environmental conditions, e.g extreme temperatures
- making vaccines
- producing medicines for treating disease
- hormones: insulin, human growth hormone, sex hormones
- enzymes for food digestion or to improve the flavour
- tomatoes: insertion of a gene that has a base sequence that is complementary to that of the gene producing the enzyme that causes tomatoes to soften. The two mRNA strands combine, preventing the mRNA of the original gene from being translated so the softening enzyme is not produced
- herbicide-resistant crops: so the herbicide kills the weeds but not the crops
- disease-resistant crops
- pest-resistant crops: crops given a gene that makes a toxin that kills insects that eat it
- plants that produce plastics
Transfer of genes from an animal that has natural resistance to a disease into a totally different animal. This second animal is then made resistant to that disease. This allows domesticated animals to be more economic to rear and hence help to reduce the price of food production.
Another example is fast growing food animals, e.g sheep that have a growth hormone gene added so they can grow larger than normal
Another example is in the production of rare and expensive proteins for use in medicine. Milk producing animals can be used. The gene for the required protein is inserted alongside a gene that codes for proteins in goats milk. The required protein is therefore produced in the milk. The gene can be inserted into the fertilised egg, so all the female offspring will be capable of producing it.
An example of this is from the genetic disorder that affects an allele that codes for the protein anti-thrombin. The gene for anti-thrombin production is added to the fertilised eggs, along with the gene that codes for proteins in goats milk. They are implanted into the goats, and bred to make goats that produce milk rich in the protein anti-thrombin. This anti-thrombin is extracted from the milk and given to humans to manufacture anti-thrombin.
Gene Therapy- Cystic Fibrosis
Cystic Fibrosis is caused by a mutant recessive allele in which three DNA bases, AAA, are missing due to a deletion mutation. The normal gene, cystic fibrosis trans-membrane-conductance regulator (CFTR) gene, normally produces a protein. However, this deletion results in an amino acid left out of the protein, so the protein is unable to perform its role of transporting chloride ions across epithelial membranes. CFTR is a chloride ion channel protein that transports chloride ions out of epithelial cells, and water naturally follows by osmosis (keeping epithelial membranes moist). The defective gene means that the protein is either not made or does not function normally. The epithelial membranes are dry and the mucus is sticky, causing:
- mucus congestion in the lungs, leading to higher risk of infection as mucus that traps disease causing organisms cannot be removed
- breathing difficulties and less efficient gaseous exchange
- accumulation of thick mucus in the pancreatic ducts, preventing pancreastic enzymes from reaching the duodenum, leading to fibrous cysts
- accumulation of thick mucus in sperm ducts, leading to infertility
Gene Therapy Treatment
Treatment of Cystic Fibrosis using gene therapy:
- gene replacement: the defective gene is replaced with a healthy gene
- gene supplementation: one or more copies of the healthy gene are added alongside the defective gene. As the added genes have dominant alleles the effects of the recessive alleles of the defective gene are masked
There are also two different techniques of gene therapy:
- germ line gene therapy: replacing or supplementing the defective gene in the fertilised egg. This ensures that all cells of the organism will develop normally, as will the cells of their offspring. This is a more permanent solution, affecting future generations. However, there are moral and ethical issues.
- somatic cell gene therapy: targets just the affected tissues, such as the lungs, and the additional gene is therefore not present in the sperm or egg cells and so is not passed on to future generations. As the cells of the lungs are continuously dying and being replaced, the treatment needs to be repeated periodically.
Delivering cloned CFTR genes
The aim of somatic-cell gene therapy is to introduce cloned normal genes into the epithelial cells of the lungs. This can be carried out in two ways:
- using a harmless virus: viruses cause colds and respiratory diseases by injecting their DNA into the epithelial cells of the lungs. They therefore make useful vectors for the transfer of the normal CFTR gene into the host cells:
- the adenoviruses are made harmless by interfering with a gene involved in their replication
- these are then grown in epithelial cells in the laboratory along with plasmids that have had the normal CFTR gene inserted
- the CFTR gene becomes incorporated into the DNA of the adenoviruses
- these adenoviruses are isolated from the epithelial cells and purified
- the adenoviruses with the CFTR gene are introduced into the nostrils of patients
- they inject their DNA, which includes the normal CFTR gene into the epithelial cells of the lungs
The second way is:
wrapping the gene in lipid molecules:
- genes are wrapped in lipid molecules because they can relatively easily pass through the phospholipid portion of the cell surface membranes
- CFTR genes are isolated from healthy human tissue and inserted into basterial plasmid vectors
- the plasmid vectors are reintroduced into their bacterial host cells and gene markers are used to detect which bacteria have successfully taken up the plasmids with the CFTR gene
- these bacteria are cloned to produce multiple copies of the plasmids with the CFTR gene
- the plasmids are extracted from the bacteria and wrapped in lipid molecules to form a liposome
- the liposomes containing the CFTR gene are sprayed into the nostrils of the patient as an aerosol and are drawn down into the lungs during inhalation
- the liposomes pass along the phospholipid portion of the cell surface membrane of the lung epithelial cells
These forms of delivery are not always effective because:
- adenoviruses may cause infections
- patients may develop immunity to adenoviruses
- the liposome aerosol may not be fine enough to pass through the tiny bronchioles of the lungs
- even where the CFTR gene is successfully delivered to the epithelial cells, very few are actually expressed.
Gene Therapy- SCID
Severe combined immunodecifiency (SCID) is a rare inherited disorder. Sufferers do not show a cell mediated immune response and cannot produce antibodies. The disorder arises when individuals inherit a defect in the gene that codes for the enzyme ADA. This enzyme destroys toxins that would kill white blood cells. There have been gene therapy attempts to treat the disorder:
- the normal ADA gene is isolated from healthy human tissue using restriction endonucleases
- the ADA gene is inserted into a retrovirus
- the retroviruses are grown with host cells in the laboratory to increase their number
- the retroviruses are mixed with the patients T cells
- the rertoviruses inject a copy of the normal ADA gene into the T cells
- the T cells are reintroduced into the patient's blood to provide the genetic code needed to make ADA
This treatment is limited because T cells live for 6-12 months so treatment has to be repeated at several intervals. More recent treatment involves transforming bone marrow stem cells rather than T cells. As bone marrow stem cells divide to produce T cells, there is a constant supply of the ADA gene.
Disadvantages of Somatic Gene Therapy
- the effects are short lived: somatic cells are not passed on to daughter cells so repeat treatments are necessary
- can induce an immune response: both the gene and the vector/liposome can induce an immune response which can result in it being rejected.
- using viral vectors to deliver gene present problems: viruses can lead to toxic, inflammatory and immune responses. Disabled viruses can also recover the ability to cause disease once inside the patient
- genes are not always expressed
- it is not effective in treating conditions that arise in more than one gene: gene therapy works best in disorders that are the result of a single mutation.
Locating and Sequencing Genes
A DNA probe is a short, single-stranded section of DNA that has some sort of label attatched that makes it identifiable:
- radioactively labelled probes: made up of nucleotides with the isotope 32P
- fluorescently labelled probes: emit light (fluoresce) under certain conditions
DNA probes are used to identify particular genes:
- a DNA probe is made that has bases that are complementary to the portion of DNA sequence that makes up part of the gene whose position we want to find
- the DNA that is being tested is treated to separate its two strands
- the separated DNA strands are mixed together with the probe, which binds to the complementary bases on one of the strands: DNA hybridisation
- the site that the probe binds to can be identified with its radioactive or flouenscence
The Sanger method is used to sequence the exact order of nucleotides in a section of DNA. It uses modified nucleotides that cannot attatch to the next base in the sequence when they are being joined together. They act as terminators, ending the synthesis of a DNA strand. The first stage is to set up 4 test tubes, each containing:
- many single stranded fragments of the DNA to be sequenced. This acts as a template for synthesis of the complementary strand
- a mixture of nucleotides with the bases A T G C
- a small quantity of one of the four terminator nucleotides (A* T* G* C*)
- a primer to start the process of DNA synthesis, this primer is radioactively or fluorescently labelled
- DNA polymerase to catalyse DNA synthesis
Depending on where the terminator nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few nucleotides or after a long fragment of DNA has been synthesised. Therefore, the DNA fragments will be of varying lengths.
The next stage is to separate out these different length fragments of DNA. This can be done through gel electrophoresis.
The DNA fragments are placed onto 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. So smaller fragments move further than the larger fragments.
Only DNA fragments up to 500 bases can be sequenced in this way, larger genes must be cut into smaller fragments by restriction endonucleases and each fragment sequenced. Restriction Mapping is used to piece these fragments back together.
Restriction mapping involves cutting DNA with a series of different restriction endonucleases. The fragments produced are separated by gel electrophoresis.
Automated restriction mapping: instead of radioactively labelling the primer, the four terminators are labelled with fluorescent die, each base takes up a different colour.
People who are heterozygous with a recessive allele for a genetic disorder will not display symptoms of the disease but will carry one copy of the mutant allele. They have the capacity to pass the disease to their offspring if the other parent is also heterozygous or homozygous recessive.
Therefore, we must screen individuals who may carry a mutant allele. Screening can determine the probability of a couple having offspring with a genetic disorder (and advice is given from a genetic counsellor).
Genetic screening can also detect oncogenes that are responsible for cancer. Cancers may develop as a result of mutations that prevent the tumour suppressor genes inhibiting cell division. Mutations of both alleles must be present to inactivate the tumour suppressor genes and to initiate the development of a tumour. Some people inherit one mutated tumour suppressor gene; these people are at greater risk of developing cancer.
If a mutated gene is detected, individuals can then make decisions about their lifestyle and future treatment. They can also check themselves more regularly for signs of cancer, leading to earlier diagnosis and better chance of successful treatment. They also may choose to have gene therapy.
This is a apsecial form of work, where advice and information are given that enable people to make personal decisions about themselves or their offspring. One important aspect of it is to research the family history of an inherited disease and to advise parents on the likelihood of it arising in their children.
The counsellor can also inform the couple of the effects of the disorder and its emotional, psychological, medical, social and economic consequences. They 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.
Genetic fingerprinting relies on the fact that the genome of any organism contains many repetitive, non-coding bases of DNA. 95% of human DNA does not code for any characteristics, and these non-coding bases are introns. They contain repetitive sequences of DNA called core sequences, which have their own unique pattern in each individual (except identical twins). The more closely related two individuals are, the more similar the core sequences will be.
The making of genetic fingerprinting consists of 5 main stages:
Extraction: separating the DNA from the rest of the cell (e.g hair or blood cell), as the amount of DNA is usually small, its quantity can be increased by using PCR
Digestion: DNA is cut into fragments using restriction endonucleases. These are chosen for their ability to cut close to groups of core sequences
Separation: fragments of DNA are next separated according to size by gel electrophoresis under the influence of an electrical voltage. The gel is immersed in alkali to separate the double strands into single strands. The single strands are then transferred on to a nylon membrane by Southern blotting, which involves a series of stages:
- a thin nylon membrane is laid over the gel
- the membrane is covered with several sheets of absorbent paper, which draws up the liquid containing the DNA by capillary action
- this transfers the DNA fragments to the nylon membrane in precisely the same positions
- the DNA fragments are then fixed to the membrane using ultraviolet light
Hybridisation: radioactive DNA probes are now used to bind with the core sequences. The probes have complementary base sequences to the core sequences and bind to them under specific conditions, e.g temperature and pH. The process is carried out with different probes, each of which binds with a different core sequence.
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 bars is revealed. The pattern of bands is unique to every individual (except identical twins).
If there appears to be a match, the patterns of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of DNA fragments from the bands. The closer the match between the two patterns, the greater the probability that the two sets of DNA have come from the same person.