- Created by: Abbie
- Created on: 26-01-20 15:36
Substitution of bases
This is where a nucleotide in a section of DNA is replaced by another nucleotide with a different base. There are three possible outcomes of this:
- one of the three stop codons is produced, meaning polypeptide creation is stopped prematurely. The final protein produced will almost certainly be significantly different, and may not perform the intended function. This is a 'non sense' mutation
- the codon that's formed codes for a different amino acid, altering the structure of part of the polypepide, so that the protein may differ in shape and not function properly. This is a 'mis sense' mutation
- degenerate genetic code, so a different codon that codes for the same amino acid could be formed. This is a 'silent' mutation
- Deletion: This is the loss of one/more nucleotide in DNA's base sequence.This results in a 'frame shift' to the left; the reading frame has been moved to the left by one (or more) letter(s), meaning that the gene is now read in the wrong 3-base groups. This alters most of the triplets, and the amino acids they code foe, meaning that the altered polypeptides could create a non-functional protein. This can considerably alter phenotype
- Addition: extra base added; usually causes a frame shift to the right, unless 3 bases (or a multiple of 3) are added. This still produces an altered polypeptide, but not to the extent a frame shift would
- Duplication: 1/more bases are repeated, producing a frame shift to the right
- Inversion: a group of bases are separated from DNA and rejoin in an inverted order, impacting the amino acid sequence
- Translocation: a group of bases separated from DNA in one chromosome and is inserted into another chromosome. This usually has a significant impact on gene expression, creating an absnormal phenotype which increases the risk of certain diseases like cancer
Although gene mutations arise spontaneously during DNA replication, they are increased by mutagenic agents:
- high energy ionising radiation- e.g. alpha and beta particles can change DNA's structure, causing problems during replication. For example, UV radiation can cause adjacent thymine bases to pair up together
- Chemicals can delete or alter bases, e.g. alkylating bases can add an alkyl group to guanine, making the structure change as it pairs with thymine (instead of cytosine)
- Base analogs are chemicals that can substitute for a base during DNA replication, changing the base sequence in the new DNA
Non-disjunction in meiosis can also cause mutations
- All cells in an organism carry the same genes, however not all genes in every cell are expressed (transcribed and used to make polypeptides). Therefore, different cells produce different proteins, depending on which genes are expressed; these proteins modify the cell, determining cell structure and controlling its processes. The transcription of genes is controlled by proteins called transcription factors. This in turn controls the expression of a gene.
- In eukaryotes, TFs move from the cyotoplasm to the nucleus, where they bind to a specific DNA site, called a promoter region, upstream in DNA sequence (5' end), near the start of the target gene.
- As the TF has a complementary tertiary structure to the promoter, when the two bind together, they're able to inititae the beginning of transcription in the gene (producing mRNA) by causing RNA polymerase to move down the strand. This 'switches' the gene on, allowing it to be expressed
- When a gene isn't being expressed, the site on the TF that binds to the DNA is inactive, preventing transcription from occuring
- TFs can be 'activators', which stimulate transcription, or 'repressors', which inhibit transcription by preventing RNA polymerase from binding to the gene
Oestrogen- initiating transcription
Oestrogen is a steriod hormone that can combine with an oestrogen receptors site on a TF in order to form an oestrogen-oestrogen receptor complex, due to the complementary shapes of the two. This stimulates the expression of genes:
- Oestrogen is lipid-soluble, so can diffise through the phospholipid bilayer and combine with the TF found in the cytoplasm
- When the two bind, the oestrogen changes the shape of the DNA binding site on the TF, making it complementary to that on the promoter, so that the gene can be activated.
- The complex enters the nucleus via a nuclear pore, binding to the binding sites on the promoter region and therefore stimulating the transcription of the gene by causing RNA polymerase to begin moving down it
An over-production of oestrogen has been linked to around 35% of breast cancers, possibly due to the over-expression of genes causing uncontrolled cell division, and therefore tumour formation. Drugs containing endoxifen have been prescribed to help this due to its ability to bind to the oestrogen receptor sites on the TF (similar structrue to oestrogen) and therefore prevent the oestrogen from binding and providing a complementary DNA binding site; silences the gene.
RNA interference- siRNA
RNA interference (RNAi) can prevent the translation of mRNA in eukaryotes through the use of RNAi molecules called siRNA (small interfering RNA) and miRNA (microRNA); these are small, double-stranded lengths of non-coding RNA (don't code for proteins) that stop target genes from being translated into proteins.
- mRNA is over-expressed and needs to be intercepted in the cytoplasm before it reaches the ribosome, in order to stop translation. A double stranded siRNA version of this mRNA is needed to silence it.
- Double-stranded RNA binds to, and is broken up by enzyme DICER into chunks of siRNA
- The siRNA binds to an argonaute protein, and the strand that is complementary to the initial mRNA strand (the 'guide strand') is selected and remains bound to argonaute. This, along with other proteins, forms a RISC complex (RNA induced silencing complex)
- The siRNA guides the RISC complex to the mRNA, which uses the siRNA strand to bind to the complementary section of the original mRNA strand, allowing argonaute to catalyse cleavage of the mRNA, which will then be degraded.
RNA interference- miRNA
miRNA also guides RISC to mRNA. Whilst siRNA usually has perfect complementarity to the mRNA, usually only part of the miRNA (called the seed) is complementary to the mRNA strand/pairs with it. Because of this lack of specificty, miRNA can target hundreds of mRNA strands, leading to them being degraded or translation inhibited. However, instead of the proteins associated with miRNA cutting mRNA into fragments, the miRNA-protein (RISC) complex physically blocks the translation of traget mRNA. The mRNA is then either stored or degraded.
Argonauts are found in plants, animals, fungi and some bacteria.
Epigenetics is a field that attempts to explain how environmental influences can impact the genetic inheritance of an organism's offspring. In eukaryotes, epigenetic control determines whether a gene is switched on/off (expression). This is achieved through the attachment/removal of chemical groups called epigenetic marks to/from DNA and histone proteins. They don't impact DNA's base sequence, but impact how easy it is for transcription proteins to transcribe DNA:
DNA and histones are covered in chemicals called 'tags'. These form a second layer called the epigenome, which determines the shape of the DNA-histone complex; e.g. it can keep inactive genes switched off by keeping them in a tightly packed arrangement so they can't be reached by transcription proteins. This is called epigentic silencing and can also work conversally to switch genes on. The epigenome is flexible, as tags respond to environmental changes which cause the chemical tags to adjust the unwrapping/wrapping of DNA to switch genes on/off.
The epigenome and development
A cell's epigenome is the accumulation of the signals it has recieved in its lifetime, so is a 'cellular memory'. When a person is in early development, signals coming from within the cells of the fetus and the nutrition provided bt the mother is very important in shaping the epigenome. Once they're born, and throughout life, environmental factors and signals within the body (like hormones) influence the epigenome. These factors can cause it to inhibit or activate specific sets of genes.
Environmental signals stimulate proteins, which carry the message along to the nucleus. Here, the message attaches to a specific protein, which can be attached to a specific sequence of bases in the DNA; the attachment of the protein can have two possible effects. It can change:
- acetylation of histones, leading to the activation or ingibition of a gene
- methylation of DNA by attracting enzymes that can add or remove methyl groups
Epigenetics can be inhertited; it's thought that in sperm and egg cells during the earliest stages of development, a specialised cellular mechanism searches the genome, removing its epigenetic tags in order to have a 'clean slate'. However, a few epigenetic tags escape this process, and pass on from parent to offspring.
Increased DNA methylation switches a gene off:
- Methylation is the addition of a methyl group (CH3), and is used to silence genes. The methyl group is an epigenetic mark that is attached to a DNA sequence by the cytosine bases.
- The group always attaches at a CpG site, with this being read from the 5' end of DNA
- Increased methylation alters DNA's structure so that the transcription factor is no longer complementary to the promoter region on the DNA, preventing the two from binding.
Decreased acetylation of histones can switch genes off:
- DNA wraps around histones to form chromatin, which makes up chromosomes. If the DNA-histone complex is highly condensed, the DNA is not accessible to transcription factors, so the gene is switched off
- Histones can be modified by the addition/removal of acetyl groups (epigenetic marks). An aceytl group is donated from acetyl CoA during acetylation (which deacetylation being the reverse). Acetyl groups are negative, so without them, the positive charge within the histones (which are already positive) increases, creating a stronger attraction to DNA's phosphate groups that creates a greater association between DNA and histones, making them inaccessible to transcription factors. Therefore, mRNA can't be produced, and the gene is switched off.
Epigenetics and disease
- Whilst epigenetics are part of normal development, they can also cause disease. This is because the alteration of epigenetic processses can cause abnormal activation/silencing of genes that has been associated with diseases such as cancer
- For example, diseased tissue taken from cancer patients had less DNA methylation that normal tissue; as DNA methylation inhibits transcription, cancer patients with less would have higher than normal gene activity (uncontrolled cell division). Sections of DNA near promoter regions don't normally have methylation, but in cancer cells these regions become highly methylated. This causes genes that should be active to switch off.
- Whilst epigenetic changes don't alter DNA's base sequence, they can increase the incidence of mutations. Active genes that normally help DNA repair (to prevent cancer) are highly methylated in cancer patients, causing these genes to be switched off; this leads to the development of cancer, as the DNA base sequences aren't repaired
As epigenetic changes activate/silence specific genes, epigenetic treatments can be used to try to counteract these changes by using drugs to inhibit certain enzymes involved in either histone acetylation or DNA methylation. But, this must be specifically targeted to cancer cells, or it could cause normal cells to become cancerous by activating transcription. Epigenetics can also be used for diagnostic tests that detect diseases in their early stages (e.g. by identifying levels of DNA methylation and histone acetylation) in an attempt to allow people to be treated earlier in order to have a better chance of recovering
Mutations in cancer
Aquired mutations can result in uncontrolled cell division if they are impacting cells that control mitosis. This results in tumour (a mass of abnormal cells) formation; cancers are tumours that invade and destroy surrounding tissue. Mutations to these cells, which control cell division, can cause cancer:
- tumour suppressor genes- can be inactivated if there's a mutation in the DNA sequence. Normally, they slow cell division by producing proteins that slow the divison and cause apoptosis. If the protein isn't produced due to a mutation, uncontrollable cell division will occur
- proto-oncogenes- their effect can be increased by DNA mutations. Usually, the gene stimulates cell division by producing proteins. Mutations can cause the gene to become overactive, resulting in an 'oncogene' that causes uncontrollable cell division. This is either due to the receptor protein on the cell surface membrane being permenantly activated or because the oncogene codes for a growth factor protein that's produced in excessive amounts.
Some cancers are due to inhertited mutations of these genes, but the majority are due to aquired mutations.
- malignant tumours are cancerous, usually growing more quickly and invading surrounding tissues. The cells don't produce adhesion molecules, so parts can break off and spread the tumour around the body via the bloodstream/lymphatic system. This process is called metastasis and forms secondary tumours.
- benign tumours are not cancerous, so usually grow slower without invading surrounding tissue. This is due to the fact they produce adhesion molecues and are surrounded by fibrous tissue to prevent them from invading other tissue. Often, they're harmless, but can cause blockages and put pressure on organs. If not removed, sometimes benign tumours can be malignant
Tumour cells look and function differently to normal cells
- irregular shape
- nucleus is larger and darker, sometimes there's more than one
- don't produce all the proteins they need for growth
- different surface-antiens
- don't respond to growth regulating processes
- more frequent division
Abnormal methylation in growth genes
Methylation is important in regulating gene expression. Abnormal DNA methylation is common in the development of a variety of tumours:
- hypermethylation in TSG occurs in its promoter region, leading it to be inactivated. This inhibits the transcription of TSG, silencing it so that it can no longer produce the proteins needed to slow down cell division- leads to tumour formation
- hypomethylation in proto-oncogenes actiavtes them to an abnormal level (producing oncogenes); this causes cell division of mutated cells to increase at a higher level, resulting in a tumour formation
Oestrogen and breast cancer
After menopause, women have a higher risk of developing breast cancer, with this being due to the fat cells of the breasts tending to produce more oestrogen after menopause. This seems to trigger breast cancer. Once the tumour appears, it further increases oestrogen concentration due to the oestrogen binding to genes and promoting transcription of the cells. Therefore, if the oestrogen binds to one that controls cell division. it will be activated, with its continued division producing a tumour. For example, it's known that oestrogen causes proto-oncogenes of cells in the breat tissue to develop into oncogenes.
Cell differentiation and specialisation
- No one cell is able to provide the optimum conditions for all functions, as different structures, enzymes and other proteins are required
- At the start of life, all cells are identical and develop individual characteristics (specialise) as the organism matures.
- Whilst all cells contain all genes, only some are expressed at any given time; some essential genes are permanently expressed in all cells, e.g. the genes coding for respiration enzymes
- Differentiated cells produce different proteins, so tend to visibily differ frim each other. These proteins are coded for by the expressed genes
- Whilst most matured cells are no longer able to develop and specialise into other cells, some retain the ability to do so. These undifferentiated cells are known as stem cells; they occur in multicellular organisms and are able to divide to become new cells, which then become specialised
- Stem cells needed to be constantly replaced, so have the ability to divide to form an identical copy of themselves in a process called self-renewal.
- All multi-cellular organisms have some form of stem cell
Stem cells orginiate from various sources in animals:
- embryonic stem cells- come from embryos in early stages of development. They can differentiate into any tyoe of cell in the initial stages of development
- Umbilical cord blood stem cells- derived from umbilical cord blood, similar to adult stem cells
- Placental stem cells- found in placenta, develop into specific types of cells
- Adult stem cells- found in the body tissues of the fetus through to adult. Specific to a certain tissue/organ where they produce cells to repair and maintain tissue
Types of stem cells
- Totipotent stem cells- found in early embryo, can differentiate into ANY type of cell. Zygotes are totipotent, as all cells are formed from them. They divide up to 4 times each (with these initial cells also being totipotent) and mature into slightly more specialised pluripotent stem cells. In other words, these are completely unspecialised cells. During development, they'll specialise, meaning that only part of their DNA will be trasnlated and proteins made
- Pluripotent stem cells- found in embroys and can differentiate into most cells. In fact, the only one that they can't develop into is the placental cells. Their development occurs after around 4/5 days, during which time, totipotent cells start to differentiate, forming a hollow ball of cells called a blastocyst. The inner cell mass will form the animal. All the cells in inner cell mass are pluripotent
- Multipotent stem cells- pluripotent stem cells soon undergo further specialisation into multipotent stem cells; these can only differentiate into a limited number of specialised cells and usually develop into cells of a particular type- e.g. bone marrow stem cells can produce any type of blood cell. Examples inc adult stem cells and umbilical cord stem cells
- Unipotent stem cells- can only differentiate into a single type of cell and are derived from mulitoptent stem cells. Found and made in adult tissue
Unipotent stem cells-cardiomyocytes
- Cardiomyoctes are heart muscle cells that make up much of the heart's tissue. It was belived that they couldn't divide to replicare in mature mammals, so therefore meaning we're unable to regenerate our own heart cells if they become damaged
- Recent research has suggested that our hearts do in fact have some regenerative capacity, meaning that it may be possible for old/damaged cardiomycotes to be replaced with new ones derived from a small supply of unipotent stem cells in the heart
- Some researchers belived this process of regeneration could be constantly occuring, but there is a debate as to how quickly it occurs. Some think its v slow, with some cardiomyoctes never being replaced in a lifetime. Others think it's faster, with every cardiomyocyte in the the heart being replaced multiple times.
Induced pluripotent stem cells (iPS cells)
iPS cells are a type of pluripotent stem cell produced from somatic cells (any type of body cells that's not sperm or egg) that're usually unipotent. The cell can be almost any body cell; it's extracted and then genetically altered in a lab to make it aquired the characteristics of an embroyonic (type of pluripotent) stem cell. This involves inducing genes and transcriptional factors within the cell that're usually silenced to express themselves, in order to aquire new characteristics.
iPS cells are v similar to embroyonic stem cells in form and function, however are not identical. Importantly, they're capable of self-renewal, so can potentially divide indefinitely. This provides an unlimited supply to replace embroyonic stem cells in medial research, overcoming many of the ethical issues associated with them
Sources of stem cells
- Adult stem cells- obtained from the body tissues of an adult, e.g. the bone marrow, using an operation. This is relatively simple, however can cause quite a lot of discomfort
- Embryonic stem cells- obtained from embyos at an early stage of development. Embryos are created in a lab using in virto fertilisation (IVF); egg cells are fertilised by sperm outside the womb. When the embroys are 4-5 days old, stem cells are removed from them and the rest of the embryo destroyed. These stem cells are pluripotent
- Induced Pluripotent Stem Cells (iPS Cells)- these are created in a lab through the re-programming of specialised adult body cells so that they become pluripotent through the expression of new transcription factors. One of the ways these transcription factors can be introduced is by infecting the adult cells with a specially-modified virus. This virus has the genes coding for the transcription factors within its DNA- these genes are passed into the adult cell's DNA upon infection, meaning that the cell is able to produce the transcription factors.
Ethics of stem cell use
- Can save lives; stem cells can be used to grow organs for people who could otherwise die waiting for transplants. Also lowers chance of rejection if the person's own stem cells are used
- Can improve quality of life; replace people's damaged cells
- Embroys created for purposes like IVF, so why not stem cells
- Adult stem cells not as suitable as embryonic stem cells- people suffer in the meantime whilst they become more suitable
- Embryos are human and deserve the same respect are adult humans
- 'Slippery slope' tp the use of older embryos and fetuses
- Using stem cells from embryos created by IVF raises ethical issues as the procedure results in the destruction of an embryo that could become a fetus
- The genome is the entire set of DNA, inc all the genes in an organism. Tech allows us to sequence the genomes of a variety of organisms; however, these methods only work on fragments of DNA. The technique used is called whole-genome shotgun (WGS) sequencing. So, to sequence the entire genome of an organism, it must be broken up into smaller pieces first- these are sequenced and then put back in order using computer algorithms to align overlapping segments, in order to get the sequence of the entire genome
- The proteome of an organism is all the proteins it can make. As simple organisms like bacteria don't have any non-coding DNA, it's quite easy to determine their proteome from the DNA sequence of their genome. Another reason that it's easier to determine their proteomes is that prokaryotic organisms often have just one circular pieces of DNA that's not associated with histones. This is very useful in medical research, as identifying antigens (often proteins) on the surface of disease-causing bacteria and viruses can help to develop vaccines against such pathogens.
- It's harder to translate the genome of eukaryotic organisms as their DNA contains large non-coding sections, as well as regulatory genes that determine which genes should be switched on/off. Therefore it's harder to translate their genome into their proteome as it's more difficult to isolate the sections of DNA that actually code for the proteins.
Sequencing methods are continuously updated
- Sequencing methods are now often automared, more cost-effective and can be used on a large scale; e.g. pyrosequencing is a new technique that can sequence around 400 million bases in a 10 hour period. Whith these newer, faster techniques, whole genomes are able to be sequenced far more quickly.
Recombinant DNA technology
- Recombinant DNA technology involves isolating and cloning genes so that a fragment of DNA is able to be transferred into a microorganism and the cloned genes used to continuously produce the desired proteins. The resulting organism is down as a transgenic/genetically modified organism (GMO).
- DNA is able to be accepted between species as the genetic code is universal and transcription/translation mechanisms (needed to make proteins) between organisms are generally very similar.
- This means DNA can be transcribed and translated within cells of the transgenic organis, and the proteins it codes for can be maunfactured in the same way that they would in the donor organism.
In order to make a protein using gene transfer and cloning, these stages are involved:
- Isolation of the DNA fragments that have the gene for the desired protein
- Insertion of the DNA fragment into a vector
- Transformation- the transfer oof DNA into suitable host cells (transgenic organism)
- Identification of the host cells that have successfully talem up the gene, using gene markers
- Growth/cloning of the population of host cells
Reteroviruses have coded genetic info in the form of RNA; however, in a host cell, they are able to synthesise DNA from RNA using enzyme reverse transcriptase. The reason that RNA is used to make the DNA is that most cells only have two copies of each gene, making it hard to obtain a DNA fragment containing the target gene. However, cells are able to contain many mRNA molecules that are complementary to one gene, making the mRNA far easier to obtain. mRNA acts as a template on which a single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase:
- mRNA is isolated from the cell and is mixed with free DNA nucleotides of reverse transcriptase
- The reverse transcriptase uses the mRNA as a template to form a new DNA strand- cDNA
- cDNA is isolated by the hydrolysis of the mRNA with an enzyme
- double stranded DNA is formed on the template of cDNA using DNA polymerase
- this double strand is the target gene
When reverse transcriptase is used to isolate a DNA sequence/gene, the mRNA template strand will be free of introns, meaning the cDNA will be too. This means that prokaryotic cells like bacteria will be able to transcribe and translate the DNA
Isolation- restriction endoculeases
- Restriction enzymes (endonucleases) are found in bacteria and recognise specific DNA sequences of 4-6 nucleotides long called recognition seuqences; they're usually palindromes. The enzyme is then able to cut the double-stranded DNA at a particular point in such a sequence called a restriction site.
- Sometimes a cut occurs between two opposite base pairs (e.g. TTAA), leaving two straight edges called blunt ends
- Other enzymes cut DNA in a staggered fashion, leaving an uneven cut in which each strand of the DNA has exposed, unpaired bases. These sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
Isolation- gene machine
New technology means that fragments of DNA can be synthesised from scratch, even without a pre-existing DNA template. Instead, a database containing the info to produce the DNA is used:
- the required sequence is designed (if it doesn't already exist)/the required sequence of base of a gene is determined from the desired protein, finding the amino acid sequence of the protein and therefore looking up the mRNA codons, so that the complementary DNA triplets can be worked out
- the desired sequence of nucleotide bases is fed into a computer and checked for biosaftey
- the computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides by adding nucleotides one by one; the first one is fixed in place by a support. As nucelotides are added, protecting groups are also added to ensure nucleotides are joined at the right places, preventing unwanted branchin
- Once the oligonucleotide of around 20 bases has been formed, it's broken off from the support and all the protecting groups removed. Multiple are joined to make a gene; this will have no introns or non-coding DNA
- The gene is replicated using the PCR, which also constructs the coplementary strand of nucleotides to form a double-stranded gene. It multiplies the gene many times to give copies
Isolation- gene machine (2)
- Sticky ends are used to insert the gene into a bacterial plasmid. This will act as a vector for the gene so it can be stored, cloned or transferred to other organisms in the future
- Genes are checked using sequencing techniques and those with any errors are rejected.
This process is advantageous, as any sequence of nucleotides can be produced in a very short time; as well as this, the genes are free of introns/non-coding DNA so are able to be transcribed and translated by prokaryotic cells
In Vitro cloning- PCR
This is an automated and efficient process that involves copying fragments of DNA without having to clone them (this is called amplification). The PCR consists of 5 main components:
- the DNA fragment to be copied/amplified
- DNA polymerase- One such enzyme called taq polymerase is used as it is able to tolerate the high temperatures, without denaturing, that're used as part of the process
- 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- containing DNA's organic bases
- thermocycler- a computer-controlled machine that precisely varies temperatres over a period of time
There are three stages to the polymerase chain reaction:
1. separation of the DNA strand; the DNA fragments, primers and DNA polymerase are placed in the thermocycler and the temperature raised to 95 degrees celcius. This breaks the hydrogen bonds between the two strands of DNA, causing them to separate
In Vitro cloning- PCR (2)
2. addition (annealing) of the primers; the mixture is cooled to 55 degrees so that the primers can join (anneal) to their complementary bases on the DNA fragment. This provides the starting sequences for DNA polymerase to begin DNA copying, as DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two separate strands from simply rejoining
3. synthesis of DNA; the temperature is increased to 72 degrees- 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 nucleotide in sequence until it reaches the end of the chain
Both separated strands are copied simultaneously, meaning that there are 2 copies of the original fragment. The process then repeats by subjecting them to the temp cycle again, resulting in 4 strands. The whole temp cycle takes around 2 mins, meaning that the PCR has revolutionised many aspects of science and medicine by providing a very quickly way to amplify DNA
- Another method of DNA amplification is in vivo cloning, which transfers the DNA fragments into a host cell using a vector. An important aspect of this involves the use of sticky ends. Sequences of DNA cut by restriction endonucleases are called recognition sites. The sticky ends that're left are complementary to those at the other side, as they were previously paired together.
- If the same endonuclease is used to cut DNA, all of the fragments produced will have ends that're complementary to each other
- Once the complementary bases of 2 sticky ends have paired up, enzyme DNA ligase is used to bind the phosphate-sugar framework of the 2 sections of DNA, uniting them. Therefore, sticky ends allow us to combine the DNA of one organism with that of any other (provided the same restriction enzyme is used)
In vivo cloning- preparation of DNA
Before the isolated DNA is able to be put into a vector, extra lengths of DNA needed to be added to it. In order for a gene to be trasncribed, RNA polymerase needs to attach to DNA's promoter region, as this is what attaches RNA polymerase and the transcription factors together so that transcription can begin. Therefore, in order to allow the fragment to transcribe mRNA and form proteins, we have to attach it to the necessary promoter region. Similarly, an appropriate terminator region needs to be added to the other end of the DNA fragment in order to signal RNA polymerase to detach from the strand at the right point to end transcription.
In vivo cloning- insertion and transformation
Insertion of DNA into a vector- once cut and modified, the DNA needs to be joined to a carrying unit called a vector; this transfers DNA into host cells, most commonly being a plasmid. Plasmids almost always contain genes for antibiotic resistance; restriction endonucleases are used at one of these resistance genes to break the plasmid loop. This will be the same endonuclease that initially cut the DNA fragment to ensure the sticky ends are complementary. DNA ligase joins the sticky ends during ligation to combine the DNA and plasmid; the plasmid now has recombinant DNA
The vector transfers the DNA fragment into host cells- 'transformation' involves the vector being transferred into the host cells. To do this, the plasmids and bacterial cells are mixed together in a medium containing calcium ions. The combination of this and a sudden temp change 'shocks' the bacterial membrane so that it becomes more permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm.
In vivo cloning- identification
Not all bacteria cells will posses the DNA fragments with the desired gene- as little as 1% will take up the plasmids when the 2 are mixed together. Some plasmids close up without incorporatong the DNA fragment, sometimes the DNA fragment ends join together to form its own plasmid.
First, we need to identify which bacterial cells have taken up the plasmid's genes for antibiotic resistance; the R-plasmid is resistant to two antibiotics- ampicillin and tetracycline.Therefore, we can test for the resistance gene that has been unaffected be the introduction of the new gene: All of the bacteria is grown on agar plates containing the antibiotic. Those that've taken up plasmids will have aquired the resistance gene and be able to survive. The dead ones haven't taken up the plasmid. We can then use marker genes to identify which of the plasmids actually contain the new gene.
In vivo cloning- marker genes
Marker genes are used to identify a second, separate gene on the plasmid that is able to be identified for some reason:
- Antibiotic resistance marker genes-use replica plating; the antibiotic gene will have been cut of for the new DNA, meaning the plasmid is no longer resistant. So, by growing the bacteria on a culture containing the anitibiotic, we can discard any that don't die and then replicate the others using the orginial plate
- Fluorescent markers- more recent method; transfers a gene from a jellyfish that produces a green fluorescent protein into the plasmid. The gene to be cloned is transplanted into the centre of the GFP gene, meaning it no longer produces GFP. Therefore, we can look under a microscope and get rid of those that do fluoresce, as they don't contain the desired gene
- Enzyme markers- the gene that produces lactase is inserted- it turns a particular colourless substrate blue. The required gene is transplanted into the gene that makes lactase, preventing it from doing so. Whe the bacterial cells are grown on the colourless substrate, those that do turn it blue can be discounted.
Advantages of in vitro cloning
- It's extremely rapid- 100 billion copies of a gene can be made within a matter of hours, a particularly valuable advantage if only a minute amount of DNA is avaliable (e.g. from crime scenes). In a forensic setting, the PCR can quickly increase the amount of DNA available so that no time is wasted before forensic analysis and matching can occur. However, this PCR method would also incease massively any other contaminating DNA found at the scene. But, it is still useful as in vivo cloning would take days/weeks longer to produce the same amount of DNA
- It does not require living cells- the only thing required is the base sequence of the DNA that requires amplification; this avoids the need for complex culturing techniques, sparing much time and effort.
Advantages of in vivo cloning
- Very useful when we want to introduce a gene into another organism- once the gene has been introduced to the vector, it can be used to deliver the transformed gene into another organism. This has applications to humans, e.g. gene therapy
- It involves almost no risk of contamination- as only genes that have been cut by the same restriction endonuclease as the plasmid can join up with it (sticky ends), preventing contaminant DNA from being taken up by the plasmid. In contrast, in vitro cloning requires a completely pure sample, as any contaminant DNA will be amplified and could create a false result
- It's very accurate- mutations are very rare, meaning that very few errors occur whilst the DNA is being copied. In vitro cloning has the issue of any errors occuring whilst copying DNA or contaminants in the sample being copied in subsequent cycles
- It cuts out specific genes- therefore is very precise, as the transformed bacteria will be able to produce many copies of a specific gene, rather than just copies of the whole DNA sequence
- Produces transformed bacteria that can be used to produce large quantities of gene products- the transformed bacteria can produce proteins for commerical or medical use, e.g. hormones like insulin that help manage conditions like diabetes.
Gene therapy-recombinant DNA application
This is a use of recombinant DNA technology that allows genes responsible for disease development to be corrected. To do this, faultly genes (alleles) are replaced or supplemented with healthy ones. The new allele introduced into cells using a variety of vectors, including plasmids, altered viruses and liposomes. They're used because DNA is negatively charged, so can't readily cross the plasma membrane.
If the disorder is caused by 2 mutated recessive alleles, then a working dominant allele is inserted in order to supplement the faulty ones. However, if it's caused by a mutated dominant allele, you can insert a DNA fragment to silence the mutated allele so it no longer works.
Somatic gene therapy alters alleles in body cells; e.g. in CF the respiratory system is impacted, so the epithelial cells lining the lungs are targeted. Somatic therapy doesn't affected sex cells, so any offspring produced could still inherit the disease.
Germ line therapy alters alleles in sex cells, so that every cell of any offspring produced from these cells will be impacted by the therapy and won't suffer from the disease. However, due to ethical issues that surround the modification of sex cells, use of germ line therapy in humans is currently illegal.
Benefits/uses of recombinant DNA technology
- Agriculture- crops can be transformed to give higher yields and be more nutritious, allowing them to reduce the risk of famine and malnutrition. They can also be transformed to have pest resistance, so that fewer pesticides are needed- reduces costs and environmental problems associated with using pesticides. E.g. Golden Rice is a transformed plant being developed to reduce Vitamin A deficiency in areas like south Asia and Africa.
- Industry- industrial processes often use enzymes, which could be produced in large quantities from transformed organisms for less money. E.g. chymosin is an enzyme used to cheese-making that is produced in the stomach of cows, but now it can be produced from transformed organisms. Means it can be made in large quantities, relatively cheapy and without killing any cows, making some cheese suitable for vegetrians
- Medicine- many drugs and vaccines are produced by transformed organisms and can be made quicky, cheaply and in large quantities. E.g. insulin is used to treat Type I diabetes and use to come from animals, however as this wasn't human insulin it didn't work quite as well and was unethical. Human insulin is now made from transformed microorganisms, using a cloned insulin gene
Concerns about recombinant DNA technology
- Agriculture- farmers might plant only one type of transformed crop (monoculture), which could make the whole crop vulnerable to the same disease as the plants are genetically identical. Environmentalistis are concerned about monoculture reducing biodiversity. Some people are also worried about crops interbreeding with wild plants to form 'superweeds' that're resistant to herbicides. Organic farmers can have their crops contaminated by wind-blown seeds from nearby genetically modified crops- can't sell their plants as organic, so lose income
- Industry- anti-globalisation activists oppose globalisation at the expense of smaller companies, so object to the fact a few large biotechnology companies control some forms of genetic engineering. As the use of the tech increases, the companies will get larger and more powerful, which make force smaller businesses out of comission. Without proper labelling, peoplr may also not have a choice whether they eat food made by genetically engineered organisms. Lastly, some consumer markets (like the EU) won't import GM food/products, which could cause economic losses to producers who would usually sell to these markets
- Medicine- companies who own genetic engineering tech may limit the uses of technologies that could be saving lives. The technology could be used unethically, e.g. to make designer babies- this is currently illegal, however.
The Humanitarian argument
Recombinant DNA technology has many potential humanitarian benefits:
- Agricultural crops can be produced that help reduce the risk of famine and malnutrition
- Transformed crops could be used to produce useful pharmaceutical products like vaccines that could make drugs available to more people- e.g. in areas where refrigiration (that's normally needed to store vaccines) isn't available
- Medicines could be produced more cheaply, so that more people can afford them
- Recombinant DNA technology has the potential to be used in gene therapy in order to treat human diseases.
- They're short-single stranded lengths of DNA with some sort of label attached to make them easily identifiable. The two most commonly used probes are radioactively labelled probes that can be identified using an x-ray film when exposed to radioactivity and flurosecently labelled probes that emit light when bound to the target DNA sequence
- A DNA probe with a complementary base sequence to the allele we're trying to find is made. The double-stranded DNA that's being tested is treated to separate its two strands. The separated strands are mixed with the probe, which binds to the complementary base sequence on one of the strands. This is DNA hybridisation; a section of DNA/RNA is combined with a complementary single-stranded DNA section. When the separated DNA strands are cooled, the complementary bases on each strand anneal to re-form the original strand- if complementary sections of DNA are present in the mixture during cooling, they're just as likely to anneal with one of the separated DNA strands.
Locating specific alleles
By locating a specific gene, we can determine if someone possesses a mutant allele that could cause a genetic disorder:
- Determine the sequence of nucleotide bases of the mutant allele we're trying to find, either using DNA sequencing techniques or referring to a genetic library to obtain the sequence.
- A fragment of DNA is produced that has a complementary base sequence to the mutant allele we're trying to locate
- Multiple copies of our DNA probe are formed using the PCR, with a marker being attached to the DNA fragment
- DNA from the person who is suspected of having the mutant allele is heated to separate the strands and then cooled in a mixture containing many of our DNA probes
- The DNA is washed clean of any unattached probes and the remaining hybridised DNA will now be fluorescently labeles with the dye attached to the probe
- The dye is detected by shining light onto the fragments, causing the dye to fluoresce- this can be seen using a special microscope
Many genetic disorders are the result of gene mutations:
- if the mutation results in a dominant allele, then all of the indiviuals will have the genetic disorder
- if the allele is recessive, it'll only impact those who are homozygous recessive. Heterozygous people will be carriers, having the cpacity to pass the genetic disorder onto any children if both parents are carriers.
- Genetic screening can determine the probabilities of a couple having offspring with a genetic disorder and obtain advice from a genetic counsellor
As hundreds of DNA probes can fit on a slide at once, we can test for many different genetic disorders simultaneously. Genetic screening can also detect any mutated tumour suppressor genes, allowing individuals to look at the likelihood of them developing cancer. People who are at a high risk can then make informed descisions about lifestyle choices and also check themselves more regularly for early signs of cancer. This can lead to an early diagnosis and more sucessful treatment.
Personalised medicine/genetic counselling
Gentic screening also allows doctors to provide advice and care based on an individual's genotype, as some people'e genes can mean that a particular drug is more/less effective in treating a condition. This can save money that'd be wasted on overprescribing drugs and can also avoid medications that could cause harm and/or false hope.
Genetic screening goes hand in hand with genetic counselling, which gives people that advice and info needed ro make descisions about themselves and their offspring. A counsellor can also inform people about the effects of a condition and its emotional, psychological, medical, social and economic consequences. On the basis of this advice, the couple can decide 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 the children will have the condition and their chances of survival.
This is a diagnostic tool widely used in forensic science and medical diagnosis which is based on the fact that the DNA of everyone (except identical twins) is unique. DNA base sequences which are non-coding are known as VNTRs. For every individual, the number and length of VNTRs has a unique pattern; the probability of two people having identical VNTR sequences is extermemly small. However, the more closely related two people are, the more similar their VNTRs will be.
Making a genetic fingerprint involves extraction- DNA is obtained and can be amplified using the PCR, digestion- DNA is cut into fragments using restriction endonucleases, separation- the fragments are separated according to size during gel electrophoresis. This gel is the immersed into an alkali to separate the double strands into single strands. hybridisation- DNA probes bins with the complementary VNTRs and development- an x-ray film is put over the nylon membrane and exposes any radiation from the radioactive probes. This reveals a series of bands, which is unique to everyone except identical twins
Interpreting GF results
DNA fingerprints from 2 samples are visually checked; if there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machinen ad calculates the lengths of the DNA fragments using the bands. Finall, the odds are calculated of someone else having an identical fingerprint.
Uses of Genetic Fingerprinting:
- It can help to determine paternity, as each band on a DNA fingerprint should have a corresponding band on one of the parents' fingerprints, due to the inheritance of DNA
- DNA is often left at crime scenes, so it can be used to determine the possibility of a person being present at the scene of the crime
- It can help in diagnosing diseases by comparing fingerprints of people with and without the disease (and with various forms of it)
- It can be used to prevent undesirable inbreeding at farms and zoos, as well as identifying which orgaisms have a desirable trait, in order to artificially select it.