3.8.4.3 Genetic fingerprinting

Genetic fingerprinting is a diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis. It is based on the fact that everyones DNA is difference (except identical twins). It relies on the fact that the genome of most eukaryotic organisms contains many repetitive non-coding bases of DNA. DNA bases which are non-coding are known as VNTRs. For everyone the number and length of VNTRs has a unique pattern. The probability of two individuals having the same VNTRs is very low.

Gel electrophoresis
This is used to separate DNA fragments according to their size. The fragments are placed on an agar gel and a voltage is applied across it. The resistance of the gel means that the larger the fragments the fess they move. In this way fragments of different lengths are separated. If fragments are labelled (e.g with radioactive DNA probes) 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 shows where the fragment is situated.


Genetic fingerprinting
This consists of 5 main stages:

• Extraction
• extract the DNA from the sample by separating it from the rest of the cell
• Digestion
• cut the DNA into fragments
• Separation
• separate the fragments by gel electrophoresis under the influence of electrical voltage
• the gel is immersed in alkali to separate the double strands into single strands
• Hybridisation
• DNA probes are used to bind with VNTRs. Different probes for different target DNA sequences
• Development
• X-ray film is put over the nylon membrane

The pattern of bars of each sample is passed through an automated scanning machine which calculates the length of DNA fragments.

Genetic relationships and variability

Genetic fingerprinting can help resolve questions of paternity. It is also useful in determining genetic variability within a population.


Forensic science
Genetic fingerprinting can establish whether a person is likely to have been at the scene of a crime.

Medical diagnosis
Genetic fingerprinting can help diagnose diseases such as Huntington's disease.


Plant and animal breeding
Genetic fingerprinting can be used to prevent undesirable inbreeding during breeding season. It can also identify plants/animals that have a particular allele of a desirable gene.

3.8.4.2 Differences in DNA between individuals of the same species can be exploited for identification and diagnosis of heritable conditions

Often human diseases are the result of a gene mutation. Recombinant DNA technology has enabled us to diagnose and treat many of these genetic disorders. We need to know where a particular DNA sequence is located. To achieve this we use DNA probes and DNA hybridisation.

DNA probes
A DNA probe is a short single stranded length of DA that has a label attached to make it more identifiable. The most common probes are:

• radioactively labelled probes
• made up of nucleotides with the isotope 32p
• identified using an X-ray film that is exposed by radioactivity
• fluorescent labelled probes
• emit light under certain conditions (e.g when the probe has bound to the target DNA sequence)

DNA probes are used to identify particular alleles of genes in the following ways:

• 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
• the double stranded DNA that is being tested is treated to separate its two strands
• the separated DNA strands are mixed with the probe which binds to the complementary base sequence on one of the strands. This is known as DNA hybridisation.
• The site at which the probe binds can be identified by the radioactivity/fluorescence that the probe emits

DNA hybridisation

This takes pace when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases. Before this can take place the two DNA strands must be separated. This is achieved by heating the DNA until its double strands separate (denaturation). When cooled the complementary bases on each strand recombine (anneal) with each other to reform the original double strand.


Locating specific alleles
It is possible to locate a specific allele of a gene using DNA probes and DNA hybridisation. E.g to establish whether an individual possesses a mutant allele that causes a particular genetic disorder:

• we must first determine the sequence of nucleotide bases of the mutant allele we are trying to locate. This can be achieved using sequencing techniques/referring to the genetic library
• a fragment of DNA is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate
• multiple copies of our DNA probe are formed using PCR
• A DNA probe is made by attaching a marker (e.g a fluorescent dye) to the DNA fragment
• DNA from the person suspected of having the mutant allele we want to locate is heated to separate its two strands
• the separated strands are cooled in a mixture containing many of our DNA probes
• if the DNA contains the mutant allele one of our probes is likely to bind to it because the probe has base sequences that are exactly complementary to those on the mutant allele
• the DNA is washed clean of any unattached probes
• the remaining hybridised DNA will now be fluorescently labelled 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

Genetic screening

If a mutation arises in a dominant allele all individuals will have the genetic disorder. it is important to screen individuals who may be carriers (heterozygous) of a mutant allele. Screening can determine the probabilities of couples having offspring with a genetic disorder. Genetic screening can also be valuable in the detection of oncogenes. Cancers may develop as a result of mutations that prevent the tumour suppressor genes inhibiting cell division.

Another advantage of genetic screening is personalised medicine. This allows doctors to provide health advice based on an individuals genotype. Furthermore, this can improve genetic counselling.

3.8.3 Using genome projects

The Human Genome Project (HGP) was an international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA, and of identifying and mapping all of the genes of the human genome.Projects to determine the entire DNA nucleotide base sequence of organisms work by mapping the DNA base sequences that make up the genes of the organism and then map these genes on the individual chromosome of that organism. In this way a complete map of all the genetic material in an organism (the genome) is obtained.

The HGP would have been impossible without the use of bioinformatics (the science of collecting and analysing complex biological data, e.g genetic codes). It uses computers to read, store, and organise biological data at a very fast rate and utilises algorithms to analyse and interpret biological data.

Determining the complete DNA base sequence of an organism uses the technique of whole-genome shotgun (WGS) sequencing. This involves researchers cutting the DNA into many small easily sequenced sections and then using computer algorithms to align overlapping segments to assemble the entire genome. Sequencing methods are continuously updated. This and increased automation of the processes involved have lead to extremely rapid sequencing of whole genomes.

One outcome of the HGP is that lots of medical advances have been made. E.g over 1.4 million SNPs (single nucleotide polymorphisms) have been found in the human genome. SNPs are single-base variations in the genome that are associated with disease/disorders. Medical screening of individuals has allowed quick identification of potential medical problems (this is good for early intervention).

The proteins a genome codes for are known as the proteome (all the proteins produced by the genome). A protein is only produced when a gene is switched on. The cellular proteome is all the proteins produced in a given type of cell at a given time under specific conditions. The complete proteome is all the proteins produced in a given organism at a given time under specific conditions.

Determining the genome and proteome of simpler organisms
We sequence the genomes of prokaryotic/single-celled eukaryotic cells to gain information to help cure disease and provide knowledge of genes that can be usefully exploited. E.g ones from organisms that can withstand extreme/toxic environmental conditions and so have potential uses in cleaning up pollutants or in manufacturing biofuels. Determining the proteome of prokaryotes is relatively easy because:

• the vast majority of prokaryotes have just one circular piece of DNA that is not histone associated
• there are none of the non-coding portions (introns) of DNA which are typical of eukaryotic cells

Knowledge of the proteome of prokaryotes has a number of applications. One is the identification of the proteins that act as antigens on the surfaces of human pathogens. These antigens can be used in vaccines against diseases caused by these pathogens. The antigens can be manufactured and then administered to people in appropriate doses. In response, memory cells are formed etc.

Determining the genome and proteome of complex organisms

The problem in complex organisms is translating knowledge of the genome into the proteome. This is because the genome of complex organisms contains many introns (non-coding genes) as well as others that have a role in regulating other genes.

3.8.2.3 Gene expression and cancer

Cancer is a group of diseases caused by damage to the genes that regulate mitosis and the cell cycle. This leads to unrestrained growth of cells and as a consequence an abnormal group of cells (a tumour) develops and constantly expands in size.

It is important to realise that not all types of tumour are cancerous. Cancerous tumours are known as malignant tumours, non-cancerous tumours are known as benign tumours). We need to know the main characteristics of them:

Benign (non-cancerous)	Malignant (cancerous)
Grow to a large size very slowly	Can also grow to a large size, but very rapidly
The cell nucleus has a normal appearance	The cell nucleus is often larger and darker due to an abundance of DNA
Cells are often specialised (differentiated)	Cells become unspecialised (de-differentiated)
Cells produce adhesion molecules that make them stuck together so they remain within the tissue from which they arise (primary tumours)	Cells do not produce adhesion molecules and so they tend to spread to other regions of the body (metastasis) (secondary tumours)
Tumours are surrounded by a capsule of dense tissue so remain as a compact structure	Tumours are not surrounded by a capsule and can therefore grow finger-like projections into surrounding tissue
Much less likely to be life threatening but can potentially disrupt the functioning of a vital organ	More likely to be life threatening as abnormal tumour tissue replaces normal tissue
Tend to have localised effects on the body	Often have systemic effects such as weight loss and fatigue
Can usually be removed by surgery	Removal usually involves radio/chemotherapy and also surgery
Rarely reoccur after treatment	More frequently reoccur after treatment

DNA analysis of tumours has shown that cancer cells are derived from a single mutant cell. Further mutation in one of the descendent cells leads to other changes that cause subsequent cells to be different from normal cells in growth and appearance. The two main types of genes that play a role in cancer are tumour suppressor genes and oncogenes. An importance difference between oncogenes and tumour suppressor genes is that while oncogenes cause cancer as a result of the activation of proto-oncogenes, tumour suppressor genes cause cancer when they are inactivated.

Oncogenes
Most oncogenes are mutations from proto-oncogenes. Proto-oncogenes stimulate a cell to divide when growth factors attach to a protein receptor on its cell-surface membrane. This activates the genes that cause the DNA to replicate and the cell to divide. If a proto-oncogene mutates into an oncogene it can become permanently activated (switched on) for two reasons:

• The receptor protein on the cell-surface membrane can be permanently activated so that cell division is switched on even in the absence of growth factors
• The oncogene may code for a growth factor that is then produced in excessive amounts stimulating excessive cell division

The result is that cells divide too rapidly and out of control resulting in either a tumour or a cancer. A few types of cancer are caused by inherited mutations of proto-oncogenes that cause the oncogene to be activated. Most cancer-causing mutations involving oncogenes are acquired not inherited.

Tumour supressor genes
Tumour suppressor genes slow down cell division (they suppress tumours/cell division), repair mistakes in DNA, and tell cells when to die (apoptosis). They have the opposite role to proto-oncogenes. A normal tumour suppressor gene maintains normal rates of cell division which prevents tumours. If a tumour suppressor gene mutates it becomes inactive/switched off. As a result the inhibition of cell division stops and cells begin to grow out of control. The mutated cells are structurally and functionally different from normal cells. Many die but those that survive can make clones of themselves and form tumours. Some cancers are caused by inherited mutations of tumour suppressor genes but most are acquired. 

Abnormal methylation of tumour suppressor genes
Abnormal DNA methylation is common in the development of a variety of tumours. The most common abnormality is hypermethylation/increased methylation. The process whereby this leads to cancer is as follows:

• Hypermethylation occurs in a specific region (a promoter region) of tumour suppressor genes
• This leads to the tumour suppressor gene being inactivated
• As a result transcription of the promoter regions of tumour suppressor genes is inhibited
• As the tumour suppressor gene normally slows the rate of cell division it's inactivation leads to increased cell division and the formation of a tumour.

Furthermore, hypomethylation/decreased methylation can occur in oncogenes where is leads it leads to their activation hence formation of tumours.

Oestrogen and breast cancer

Oestrogen plays an important role in regulating the menstrual cycle in women. The fat cells of breasts tend to produce more oestrogen after menopause. These locally [produced oestrogens appear to trigger breast cancer in postmenopausal women. Once a tumour has developed it further increases oestrogen concentration which therefore leads to increased tumour development. White blood cells that are drawn to the tumour also increase oestrogen production. Basically how this works is, if oestrogen binds to a gene that controls cell division and growth then the gene will be activated and its continued division could produce a tumour.  It is also known that oestrogen causes proto-oncogenes to develop into oncogenes which leads to the development of a tumour.

3.8.2.2 Regulation of transcription and translation

In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus. The general principles involved in controlling gene expression by controlling transcription are as follows:

• For transcription to begin the gene must be switched on by specific molecules (transcriptional factors) that move from the cytoplasm into the nucleus
• Each transcriptional factor has a site that binds to a specific bas sequence of the DNA in the nucleus
• When it binds it causes this region of DNA to begin the process of transcription
• mRNA is produced and the information it carries is translated into a polypeptide (this is translation)
• When a gene is not being expressed (it is switched off), the site on the transcriptional factor that binds to DNA is not active
• As the site is inactive it cannot cause transcription and polypeptide synthesis.

So we need to know a bit about the role of the steroid hormone oestrogen in initiating transcription. Well, hormones like oestrogen can switch on a gene and start transcription by combining with a receptor site on the transcriptional factor. This then activates the DNA binding site on the transcriptional factor (by causing it to change shape):

• Oestrogen, a lipid-soluble molecule, diffuses easily through the phospholipid bilayer of cell-surface membranes
• Once inside the cytoplasm oestrogen binds with a site on a receptor molecule of the transcriptional factor. They are complimentary to one another
• By binding with the site the oestrogen changes the shape of the DNA binding site on the transcriptional factor, activating it
• It can now bind to DNA
• The transcriptional factor enters the nucleus (through a nuclear pore) and binds to a specific base sequence of DNA
• This stimulates transcription of the gene that makes up that portion of DNA

Whilst genes determine the features of an organism, the environment can influence the expression of these genes. It is now believed that environmental factors can cause heritable changes in gene function without changing the base sequence of DNA. This process is known as epigenetics. This provides explanations as to how environmental influences such as diets, stress, toxins (etc), can alter the genetic inheritance of an organism's offspring. So, now about how it works. Basically, we already know that DNA is wrapped around proteins called histones. We now know that both the DNA and histones are covered in chemicals (tags). These chemicals/tags form the epigenome. The epigenome determines the shape of the DNA-histone complex. E.g it keeps genes that are inactive tightly packed in arrangement ensuring they cannot be read (epigenetic silencing). It can also unwrap genes so the DNA is exposed and can be easily transcribed (switching on these particular genes). Unlike DNA, the epigenome is not fixed (it is flexible). It is flexible because its chemical tags respond to environmental changes, factors such as stress and diet can cause the chemical tags to adjust the wrapping/unwrapping, switching genes on/off.

The epigenome of a cell is an accumulation of the signals it has received during its lifetime. It acts a bit like a cellular memory. In early development the signals come from within the cells of the foetus. The nutrition provided by the mother is important in shaping the epigenome at this stage (this is why it is imperative that pregnant woman keep a good diet and don't smoke etc).After birth environmental factors affect the epigenome (although signals, such as hormones, from within the body can still influence it). These factors cause the epigenome to activate/inhibit a specific set of genes. The environmental signal stimulates proteins to carry its message inside the cell from where it is passed by a series of other proteins into the nucleus. Here the message passes to a specific protein which can be attached to a specific sequence of bases on the DNA. Once attached the protein can change:

• acetylation of histones, leading to the activation/inhibition of a gene
• methylation of DNA by attractive enzymes that can add/remove methyl groups.

Okaaay so what does any of that actually mean. Well, when the association of histones with DNA is weak the DNA-histone complex is less condensed meaning that the DNA is accessible by transcriptional factors which can initiate transcription (basically, the gene is switched on). When the association is stronger, the reverse occurs and the gene is switched off. Condensation of the DNA histone complex inhibits transcription. It can be brought about by decreased acetylation of the histones or by methylation of DNA. So how do these processes work?

Decreased acetylation of associated histones
Acetylation is the process whereby an acetyl group is transferred to a molecule. In this case, acetylcoenzyme A donates an acetyl group. Deacetylation is the removal of an acetyl group from a molecule. Decreased acetylation increase the positive charges on histones as acetyl groups are negatively charged. This increases the attraction of the histones to the phosphate groups of DNA. The association between DNA and histones becomes stronger and the genes are switched off.

Increased methylation of DNA
Methylation is the addition of a methyl group to a molecule. In this case a methyl group is added to the cytosine bases of the DNA. It inhibits transcription in the following ways:

• prevents the binding of transcriptional factors to DNA
• attracts proteins that condense the DNA-histone complex by inducing the deacetylation of histones.

Epigenetic changes can be responsible for certain diseases. Altering the epigenetic process can cause abnormal activation/silencing of a gene. In some cases the activation of a normally inactive gene can cause cancer. In other cases, the inactivation (silencing) of a usually active gene causes a disease.

In specific sections of DNA (near promoter regions) that have no methylation in normal cells. In cancer cells these regions become highly methylated causing genes that should be active to switch off.

Whilst epigenetics do not alter the sequence of bases in a DNA molecule they can increase the incidence of mutations. For example, some active genes help to repair DNA (preventing cancers). Individuals with various types of inherited cancer have increased methylation of these genes causing the gene to be switched off. As a result, the damage to base sequences in DNA are not repaired. This can lead to the development of cancer.

It's not all bad news though, we can also use epigenetic treatments to counteract the epigenetic changes that cause certain genes to be activated/silenced. The treatments use drugs to inhibit certain enzymes involved in either histone acetylation or DNA methylation. E.g. drugs that inhibit enzymes that cause DNA methylation can reactivate silenced genes. 

Epigenetics have also been used in diagnostics tests to detect the early stages of diseases such as cancer/brain disorders/arthritis. The tests can identify the level of DNA methylation and histone acetylation at an early stage of disease. This allows patients to seek treatment asap.

RNA interference
This is the last little bit in this section. Basically, in eukaryotes (and some prokaryotes!) the translation of mRNA produced by a gene can be inhibited by breaking mRNA down before its coded information can be translated into a polypeptide.One type of RNA molecule that may be involved is small interfering RNA (siRNA). This mechanism involved small double-stranded sections of siRNA and operates as follows:

• An enzyme cuts large double stranded RNA molecules into smaller sections known as siRNA
• One of the two siRNA strands combines with an enzyme
• The siRNA molecule guides the enzyme to a mRNA molecule by pairing up its bases with the complimentary ones on a section of the mRNA molecule
• Once in position the enzyme cuts the mRNA into smaller sections
• the mRNA is no longer capable of being translated into a polypeptide meaning that the gene has not beed expressed (so it has been silenced/blocked)

3.8.2.1 Most of a cell’s DNA is not translated

In multicellular organisms cells are specialized to perform specific functions. For example red blood cells carry oxygen whilst phagocytes carry out phagocytosis. The process by which cells develop into a specialized structure suited to their function is cell differentiation. In early development all cells are the same. As the organism matures it adapts to the function it will perform when it's mature.

All multicellular cells are derived by mitotic divisions f the zygote (fertilised egg) meaning they all contain the same genes. However, only certain genes are expressed in any certain cell at a time. Certain genes are always expressed such as genes that code for enzymes required for respiration.

As mentioned above, all cells develop from a single fertilised egg. Cells which can differentiate into any body cell (such as fertilised eggs) are known as totipotent cells. In mature mammals very few cells retain the ability to differentiate into any cell - these are known as stem cells. These are undifferentiated dividing cells that must be constantly replaced - they have the ability to divide and form an identical copy of themselves (self-renewal). There are a variety of sources of stem cells in mammals:

• Embryonic stem cells
• Come from embryos in the early stages of development. Can differentiate into any type of cell
• Umbilical cord blood stem cells
• Are derived from umbilical cord blood and are similar to adult stem cells
• Placental stem cells
• Found in the placenta and develop into specific types of cells
• Adult stem cells
• Found in the body tissues of the fetus through to the adult. Are specific to a particular tissue/organ within which they produce the cells to maintain and repair tissues throughout an organism's life

There are a few different types of stem cell we need to know about:

• Totipotent stem cells
• Found in the early embryo
• Can differentiate into any type of cell
• Pluripotent stem cells
• Found in embryos
• Can differentiate into almost any type of cell
• Examples include embryonic stem cells and fetal stem cells
• Multipotent stem cells
• Found in adults
• Can differentiate into a limited number of specialized cells - usually of a particular type (e.g stem cells in bone marrow can poduce any type of blood cell)
• Examples include adult stem cells and umbilical cord lood stem cells
• Unipotent stem cells
• Can only differentiate into a single type of cell
• Are derived from multipotent stem cells and are made in adult tissue
• Examples include cardiomyocytes (muscle cells that make up the cardiac muscle)

Induced pluripotent stem cells (iPS cells)

These are a type of pluripotent cell that is produced from unipotent stem cells. The unipotent stem cell may be almost any body cell. It is then genetically altered to make it acquire the characteristics of embryonic stem cells. To make it aquire the charateristics we induce genes andtranscriptional factors. 

iPS cells are usually not exact copies of embryonic tem cells. They are capacble of self-renewal meaning that potentially they can divide to produce an unlimited supply.

Pluripotent cells in treating human disorders

Pluripotent cells have endless uses. they can be used to treat...

• Burns and wounds (skin cells)
• Heart damage (cardiac muscle cells)
• Muscular dystrophy (skeletal muscle cells)
• Type 1 diabetes (beta cells of the pancreas)
• Parkinsons disease/multiple sclerosis/strokes/Alzheimer's disease/paralysis due to spinal injury (nerve cells)
• Leukaemia/inherited blood diseases (blood cells)
• Osteoperosis (bone cells)
• Osteoarthritis (cartilage cells)
• Macular degeneration (Retina cells of the eye)

3.8.1 Alteration of the sequence of bases in DNA can alter the structure of proteins

When DNA replicates there is a chance of gene mutation. There are 6 types of mutation we need to know...

• Addition
• Insertion of an extra base. This results in a frame shift meaning that most triplets and therefore amino acids will be different - unless 3 bases are added.
• Deletion
• This is the loss of a nucleotide. This causes a frame shift meaning most triplets and therefore amino acids will be different. The polypeptides will therefore be different and a non-functioning protein will most likely result. A deletion at the end of a sequence is likely to have less impact.
• Substitution
• A nucleotide is replaced with (substituted by) another nucleotide. This could...
• Form a stop codon (very bad as polypeptide production would stop for the polypeptide being produced creating an incomplete protein)
• Form a different amino acid (the protein may differ in shape and therefore not function properly. This leads to conditions such as sickle cell anaemia
• The formation of a different codon which produces the same amino acid due to the degenerate genetic code.
• Inversion
• A group of bases separates from the DNA and rejoins in the same position but back-to-front
• Duplication
• One or more bases are duplicated. This has a similar affect to addition mutation therefore produces a frame shift
• Translocation of bases
• A group of bases separates from the DNA sequence on one chromosome and becomes inserted to a DNA sequence on a different chromosome. This often leads to an abnormal phenotype such as reduced fertility and development of certain cancers.

Causes of mutations

Mutations can arise spontaneously during DNA replication. This random and spontaneous occurrence can increase by outside factors known as mutagenic agents (mutagens) including:

• Chemicals
• For example nitrous oxide can directly alter the structure of DNA/interfere with transcription and bemnzopyrene (in tobacco smoke) can inactivate a tumour suppressor gene (TP53) leading to cancer.
• High energy ionizing radiation
• For example, alpha and beta particles and short length radiation such as X-rays and UV light. These can disrupt the structure of DNA.