The control of gene expression Flashcards
Genetic mutations
- Insertion
- Causes a frameshift
- When a nucleotide is randomly
inserted into DNA sequence - Affects function of polypeptide
Cause Huntington’s Disease
Genetic mutations
- Substitution
- Where a DNA base/ nucleotide is swapped for a different one
- Silent substitution alters amino acid sequence
- Missense substitution alters single amino acid
- Nonsense substitution creates a premature stop codon
Cause Sickle Cell Anemia
Genetic mutations
- Deletion
- Causes a frameshift
- A nucleotide is randomly deleted
- Affects function of polypeptide
Cause Cystic Fibrosis
Genetic mutations
- Inversion
- A single gene is cut into 2 pieces, inverted 180° and rejoined
- Results in non functional protein
Cause Haemophilia A
Genetic mutations
- Duplication
- One or more bases are duplicated in the DNA sequence
- Original gene is not changed
Cause Charcot- Marte Tooth Disease
Genetic mutations
- Translocation
- A section of a chromosome is added to another chromosome which is not its homologous partner
- Philadelphia chromosome (22) found in Leukemia Cancer
Cause Cancer, Infertility and Down Syndrome
Mutagenic agents / Mutagens
- High energy ionising radiation for example, short wavelength radiation such as X-rays and ultra violet light can disrupt the structure of DNA.
- Chemicals such as nitrogen dioxide may directly alter the structure of DNA or interfere with transcription.
(Benzopyrene, a consitituent of tobacco smoke, is a powerful mutagen that inactivates a tumour-suppressor gene TP53 leading to a cancer)
Cell differentiation
When a cell becomes specialised through differential gene expression to carry out a particular function
What is pluripotency?
Pluripotent stem cells are found in embryos and can differentiate into almost any type of cell.
- Examples are embryonic stem cells and fetal stem cells.
What is unipotency?
Unipotent stem cells can only differentiate into a single type of cell.
- They are derived from multipotent stem cells and are made in adult tissue.
What is multipotency?
Multipotent stem cells are found in adults and can differentiate into a limited number of specialised cells.
- Examples of multipotent cells are adult stem cells and umbilical cord blood stem cells
What is totipotency?
Totipotent stem cells are found in the early embryo and can differentiate into any type of cell.
- Since all body cells are formed from a zygote, it follows that the zygote is totipotent, as it divides and matures, its cells develop into slightly more specialised cells called pluripotent stem cells.
Induced pluripotent stem cells (iPS cells)
Produced from adult somatic cells using appropriate protein transcription factors
Embryonic stem cells
Come from embryos in the early stages of development
- totipotent if taken in the first 3-4 days after fertilisation
- pluripotent if taken on day 5
Adult stem cells
Found in the body tissues of the fetus through to the adult.
They are specific to a particular tissue or organ within which they produce the cells to maintain and repair tissues throughout an organism’s life.
- multipotent
- unipotent
Umbilical cord blood stem cells
Derived from umbilical cord blood and are similar to adult stem cells
Placenta stem cells
Found in the placenta and develop into specific types of cells
What are stem cells?
Stem cells are undifferentiated dividing cells that occur in adult animal tissues and need to be constantly replaced. They therefore have the ability to divide to form an identical copy of themselves in a process called self-renewal.
- Each transcriptional factor has a site that binds to a specific base sequence of the DNA in the nucleus.
- When it binds, it causes this region of DNA to begin the process of transcription.
- Messenger RNA (mRNA) is produced and the information it carries is then translated into a polypeptide.
- When a gene is not being expressed (i.e is switched off) the site on the transcriptional factor that binds to DNA is not active.
- As the site on the transcriptional factor binding to DNA is inactive it cannot cause transcription and polypeptide synthesis.
What are transcriptional factors?
For transcription to begin the gene is switched on by specific molecules (transcriptional factors) that move from the cytoplasm into the nucleus.
Oestrogen
A steroid hormone involved in switching on a gene and thus starting transcription by combining with a receptor site on the transcriptional factor, this activates the DNA binding site by causing it to change shape.
The effect of oestrogen on gene transcription
- Oestrogen is a lipid-soluble molecule and therefore diffuses easily through the phospholipid portion of cell-surface membranes
- Once inside the cytoplasm of a cell, oestrogen binds with a site on a receptor molecule of the transcriptional factor. The shape of this site and the shape of the oestrogen molecule complement one another
- By binding with the site, oestrogen changes the shape of the DNA binding site on the transcriptional factor, which can now bind to DNA (it is activated)
- The transcriptional factor can now enter the nucleus chrough a nuclear pore and bind to specific base sequences on DNA
- The combination of the transcriptional factor with DNA stimulates transcription of the gene that makes up the portion of DNA.
What are epigenetics?
A relatively new scientific field that provides explanations as to how environmental influences such as diet, stress, toxins, etc can subtly alter the genetic inheritance of an organism’s offspring
What chemicals cover DNA and histones
Known as tags
The epigenome
All of the chemical modifications to all histone proteins and DNA (except base changes) in an organism, determining the shape of the DNA-histone complex
- it is flexible as the chemical tags are influenced by the environment
- the accumulation of the signals it has received during its lifetime and it therefore acts like a cellular memory
How does the epigenome determine the DNA-histone complex shape?
It keeps genes that are inactive in a tightly packed arrangement and therefore ensures that they cannot be read, switches them off (epigenetic silencing)
It unwraps active genes so that the DNA is exposed and can easily be transcribed, switching them on
How do hormones influence the epigenome?
Activate or inhibit specific sets of genes.
How does the environment influence the epigenome?
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 or bases on the DNA. Once attached the protein has two possible effects. It can change:
- acetylation of histones leading to the activation or inhibition a gene
- methylation of DNA by attracting enzymes that can add or remove methyl groups.
Acetylation
A chemical reaction in which a small molecule called an acetyl group is added to other molecules
Methylation
A chemical reaction in the body in which a small molecule, a methyl group gets added to DNA, proteins or other molecules
Weak association of histones to DNA
The DNA-histone complex is less condensed (loosely packed). In this condition the DNA is accessible by transcription factors, which can initiate produtction of mRNA and can switch the gene on.
Strong association of histones to DNA
The DNA- histone complex is more condensed (tightly packed). In this condition the DNA is not accessible by transcription factors, which therefore cannot initiate production of mRNA and so the gene is switched off
Correlation of association between DNA and histones
Condensation of the DNA-histone complex therefore inhibits transcription, which can be brought about by decreased acetylation of the histones or by methylation of DNA.
Deacetylation
The reverse reaction where an acetyl group is removed from a molecule.
Decreased acetylation of associated histones
Decreased acetylation increases the positive charges on histones and therefore increases their attraction to the phosphate groups of DNA.
- The association between DNA and histones is stronger and the DNA is not accessible to transcription factors. These transcription factors cannot initiate mRNA production from DNA, and so the gene is switched off.
Increased methylation of DNA
Methyl group is added to the cytosine bases of DNA. Methylation normally inhibits the transcription of genes in two ways:
- preventing the binding of transcriptional factors to the DNA
- attacts proteins that condense the DNA-histone complex (by inducing deacetylation of the histones) making the DNA inaccessible to transcription factors
Epigenetics and inheritance
It is thought that in sperm and eggs during the earliest stages of development a specialised cellular mechanism searches the genome and erases its epigenetic tags in order to return the cells to a genetic ‘clean slate’.
However, a few epigenetic tags escape this process and pass unchanged from parent to offspring.
Experiments on epigenetics and inheritance
Experiments on rats have shown that female offspring who received good care when young, respond better to stress in later life and themselves nurture their offspring better.
Female offspring receiving low-quality care, nurture their offspring less well.
- Good maternal behaviour in rats transmits epigenetic information onto their offspring’s DNA without passing through an egg or sperm.
Epigenetics and disease
- mutations
- Epigenetic changes do not alter the sequence of bases in DNA. They can increase the incidence of mutations.
- Some active genes normally help repair DNA and so prevent cancers. In people with various types of inherited cancer, it is found that increased methylation of these genes has led to these protective genes being switched off. As a result, damaged base sequences in DNA are not repaired and so can lead to cancer.
Epigenetics and disease
- methylation
- In 1983, researchers found that diseased tissue taken from patients with colorectal cancer had less DNA methylation than normal tissue from the same patients. As we saw earlier, increased DNA methylation normally inhibits transcription (switches off genes). This means that these patients with less DNA methylation would have higher than normal gene activity - more genes were turned on.
Epigenetics and disease
- methylation
- It is known that there are specific sections or DNA (ones near regions called promoter regions) that have no methylation in normal cells. However, in cancer cells these regions become highly methylated causing genes that should be active to switch off. This abnormality happens early in the development or cancer.
Treating diseases with epigenetic therapy
These treatments use drugs to inhibit certain enzymes involved in either histone acetylation or DNA methylation.
- Epigenetic therapy must be specifically targeted on cancer cells. If the drugs were to affect normal cells they could activate gene transcription and make them cancerous, so causing the very disorder they were designed to cure.
Examples of treating diseases with epigenetic therapy
- Drugs that inhibit enzymes that cause DNA methylation can reactivate genes that have been silenced.
- Development of diagnostic tests that help to detect the early stages of diseases such as cancer, brain disorders and arthritis, which can identify the level of DNA methylation and histone acetylation at an early stage of disease allowing those with these diseases to seek early treatment and so have a better chance of cure.
The effect of RNA interference on gene expression
- An enzyme cuts large double-stranded molecules of RNA into smaller sections siRNA
- One of the two siRNA strands combines with an enzyme
- The siRNA molecule guides the enzyme to a messenger RNA molecule by pairing up its bases with the complementary ones on a section of the mRNA molecule
- Once in position, the enzyme cuts the mRNA inro smaller sections
- The mRNA is no longer capable of being translated into a polypeptide.
- This means that the gene has not been expressed but it has been blocked.
Small interfering RNA (siRNA)
Small, double-stranded RNA molecules
- bind to mRNA that has been transcribed from target genes (the genes to be ‘silenced’) as their base sequence is complementary
- Each siRNA is attached to a protein complex which is able to breakdown the mRNA that has been transcribed from target genes
What is 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. As a consequence, a group of abnormal cells, called a tumour, develops and constantly expands in size.
Malignant tumours
Cancerous tumours
Benign tumours
Non- cancerous tumours
Characteristics of benign tumours
Can grow to a large size
Grow very slowly
The cell nucleus has a relatively normal appearance
Cells are often well differentiated (specialised]
Cells produce adhesion molecules that make them stick together and so they remain within the tissue from which they arise = primary tumours
Tumours are surrounded by a capsule of dense tissue and so remain as a compact structure
Much less likely to be life-threatening but can disrupt functioning of a vital organ
Tend to have localised effects on the body Can usually be removed by surgery alone Rarely reoccur after treatment
Characteristics of malignant tumours
Can also grow to a large size
Grow rapidly
The cell nucleus is often larger and appears darker due to an abundance of DNA
Cells become de-differentiated (unspecialised)
Cells do not produce adhesion molecules and so they tend to spread to other regions of the body, a process called metastasis, forming secondary tumours
Tumours are not surrounded by a capsule and so can grow finger-like projections into the surrounding tissue
More likely to be life-threatening, as abnormal tumour tissue replaces normal tissue
Often have systemic [whole body) effects such as weight loss and fatigue
Removal usually involves radiotherapy and/or chemotherapy as well as surgery More frequently reoccur after treatment
The two main types of genes that play a role in cancer
- Tumour suppressor genes
- oncogenes
Cancer and the genetic control of cell division
- generality of cancer origins
DNA analysis of tumours has shown that, in general, cancer cells arc derived from a single mutant cell. The initial mutation causes uncontrolled mitosis in this cell. Later, a further mutation in one of the descendant cells leads to other changes that cause subsequent cells to be difrerent from normal in growth and appearance
Cancer and the genetic control of cell division
- Oncogenes
Most oncogenes are mutations of proto-oncogenes which stimulate a cell to divide when growth factors attach to a protein receptor on its cell-surrace membrane. This then activates genes that cause DNA to replicate and the cell to divide. If a proto-oncogene mutates into an on cogene it can become permanently activated 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, again stimulating excessive cell division.
Cancer and the genetic control of cell division
- Tumour suppressor genes
Slow down cell division, repair mistakes in DNA, and ‘tell’ cells when to die - a process called apoptosis (programmed cell death).
They therefore have the opposite role from proto-oncogenes. A normal tumour suppressor gene maintains normal rates of cell division and so prevents the formation of tumours. If a tumour suppressor gene becomes mutated it is inactivated (switched off). As a result, it stops inhibiting cell division and cells can grow out of control. The mutated cells that are formed are usually structurally and functionally different from normal cells. While most of these die, those that survive can make clones of themselves and form tumours.
- TP53, BRCA 1 and BRCA 2
Cancer and the genetic control of cell division
- Difference between oncogenes and tumour supressor genes
While oncogenes cause cancer as a result of the activation of proto-oncogenes, tumour suppressor genes cause cancer when they are inactivated.
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:
- Hypermethylation occurs in a specific region (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.
- The inactivation leads to increased cell division and the formation of a tumour. Thought to occur in BRCA 1leading to the development of breast cancer.
Another form of abnormal merhylation is hypomethylation:
- This has been found to occur in oncogenes where it leads to their activation and hence the formation of tumours.
Oestrogen concentrations and breast cancer
- Oestrogens play a central role in regulating the menstrual cycle in women. It is known that after the menopause. a woman’s risk of developing breast cancer increases. This is thought to be due to increased oestrogen concentrations.
- At first this seemed paradoxical because the produaion of oestrogens from the ovaries diminishes after the menopause. However, the fat cells of the breasts tend to produce more oestrogens after the 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 development of the tumour.
- It also appears that white blood cells that are drawn to the tumour increase oestrogen prodution. This leads to even greater development of the tumour.
How can oestrogen cause a tumour to develop?
The mechanism by which oestrogen effectively activates a gene by binding to a gene which promotes transcription.
- If the gene that oestrogen acts on is one that controls cell division and growth, then it will be activated and its continued division could produce a tumour. It is known, for example, that oestrogen causes proto oncogenes of cells in breast tissue to develop into oncogenes. This leads to the development of a tumour (breast cancer).
What is bioinformatics
The science of collecting and analysing complex biological data such as genetic codes. It uses computers to read, store, and organise biological data at a much faster rate than previously. It also utilises algorithms to analyse and interpret biological data.
Possibilities created using bioinformatics
When you consider thar the human genome consists of over 3 billion base pairs organised into a round 20 000 genes, sequencing every one of those bases is a mammoth task and yet it took just 13 years to complete using bioinformatics
How to determine the complete DNA base sequence
The technique of whole-genome shotgun (WGS) sequencing
- researchers cut the DNA into many small, easily sequenced sections and then using computer algorithms to align overlapping segments to assemble the entire genome
What are single nucleotide polymorphisms?
SNPs are single-base variations in the genome that are associated with disease and other disorders.
Medical advancements of DNA sequencing
- Over 1.4 million single nucleotide polymorphisms (SNPs) have been found in the human genome.
- Medical screening of individuals has allowed quick identification of potential medical problems and for early intervention to treat them
- Possibility of establishing the evolutionary links between species.
What is the proteome?
The full range of proteins that a cell produces/ DNA can code for in a given time, under specified conditions
Determining the genome and proteome of simpler organisms
Used in bacteria because:
- the vast majority of prokaryotes have just one, circular piece of DNA that is not associated with histoncs
- there are none of the non-coding portions of DNA which are typical of eukaryotic cells.
First bacterium genome fully sequenced
Haemophilus influenza in 1995.
H. influenza contains 1700 genes comprising 1.8 million bases.
What is the Human Microbiome Project?
The sequencing of genomes of thousands of prokaryotic and single-celled eukaryotic organisms
- It is hoped that the information gained will help cure disease and provide knowledge of genes that can be usefully exploited
Example of DNA sequencing
P/asmodium falciparum which causes malaria.
- All 5300 genes on Plasmodium ‘s 14 chromosomes have been sequenced giving us an insight into its metabolism and knowledge of the proteins it produces.
- All this will be invaluable in helping us to develop the elusive vaccine against this globally important disease.
Determining the genome and proteome of complex organisms
- The success in mapping the human genome in 2003 is a testimony to what can be achieved in mapping DNA sequences 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 non -coding genes as well as others that have a role in regulating other genes.
There is a human proteome project currently underway to identify all the proteins produced by humans
How many genes are said to be in the human genome?
There are around 20,000 genes in the human genome although this number is constantly being revised down as our techniques for identifying genes improves
Percentage of genes which code for proteins
it is thought that as few as l.5% of genes may code for proteins
Development of recombinant DNA technology
Perhaps the most significant scientific advance in recent years
- allows genes to be manipulated, altered and transferred from organism to organism, even to transform DNA itself
Recombinant DNA technology
- isolation of the DNA fragments that have the gene for the desired protein
- insertion of the DNA fragment into a vector
- transformation, that is, the transfer of DNA into suitable host cells
- identification of the host cells that have successfully taken up the gene by use of gene markers
- growth/cloning of the population of host cells.
Several methods of producing DNA fragments
- conversion of mRNA to cDNA using reverse transcriptase
- using restriction endonucleascs to cut fragments containing the desired gene from DNA
- creating the gene in a gene machine, usually based on a known protein structure.
Resulting organism of recombinant DNA
Transgenic or genetically modified organism (GMO
What does reverse transcriptease do?
it catalyses the production of DNA from RNA.
Several methods of producing DNA fragments
- reverse transcriptease
- A cell that readily produces the protein is selected
- These cells have large quantities of the relevant mRNA, which is therefore more easily extracted
- Reverse transcriptase is then used to make DNA from RNA. (cDNA) because it is made up of the nucleotides that are complementary to the mRNA.
- To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complement ry nucleotides on the cDNA template. This double strand of DNA is the required gene
What is cDNA?
complementary DNA
Several methods of producing DNA fragments
- using restriction endonucleases
There are many types of restriction endonucleascs. Each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence.
- sometimes, this cut occurs between two opposite base pairs, leaving two straight edges known as blunt ends
- others cut DNA in a staggered fashion, leaving an uneven cut in which each strand of the DNA has exposed, unpaired bases.
Several methods of producing DNA fragments
- the ‘gene machine’
- The desired sequence or nucleotide bases of a gene is determined from the desired protein that we wish to produce.
- From this, the mRNA codons are looked up and the complementary DNA triplets are worked out.
- The desired sequence or nucleotide bases for the gene is fed into a computer.
- The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements.
- The computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene.
- In an automated process, each or the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence.
- The oligonucleotides are then joined together to make a gene. This gene doesn’t have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction
- The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double stranded gene. It then multiples this gene many times to give numerous copies.
- Using sticky ends the gene can then be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organism in the future.
- The genes are checked using standard sequencing techniques and those with errors are rejected.
The ‘gene machine’
- Advantages
Any sequence of nucleotides can be produced, in a very short time (as little as 10 days) and with great accuracy.
These artificial genes are also free of introns, and other ·non-coding’ DNA, so can be transcribed and translated by prokaryotic cells.
Gene cloning
- In vivo
By transferring the fragments to a host cell using a vector
Gene cloning
- In vitro
Using the polymerase chain reaction
In vivo
- Preparing the DNA fragment for insertion
The addition of extra lengths of DNA.
- RNA polymerase must attach to the DNA near a gene. The binding site is a promoter region, the nucleotide bases of the promoter attach both RNA polymerase and transcription factors
In vivo
- Ending the transcription process
a terminator region is added to the other end of our DNA fragment to stop transcription at the appropriate point releasing RNA polymerase
In vivo
- Insertion of DNA fragment into a vector
Fragment of DNA is joint to carrying unit, known as a vector.
This is used to transport the DNA into the host cell.
Polymerase chain reaction (PCR)
A method of copying fragments of DNA.
- The process is automated, making it both rapid and efficient.
Requirements of PCR
- the DNA fragment to be copied
- DNA polymerase
- Primers
- Nucleotides
- Thermocycles
Process of PCR
separation of the DNA strand
addition (annealing) of the primers
synthesis of DNA
The advantages of in vitro gene cloning
- Extremely rapid
- It does not require living cells
The advantages of in vivo gene cloning
- It is particularly useful where we wish to introduce a gene into another organism
- It involves almost no risk of contamination
- very accurate
- It cuts out specific genes
- Produces transformed bacteria that can be used to produce large quantities of gene product
What is a DNA probe?
A short, single-stranded length of DNA that has some sort of label attached that makes it easily identifiable.
Types of DNA probes
Radioactively labelled probes, which are made up of nucleotides with the isotope 32 P. The probe is identified using an X-ray film that is exposed by radioactivity. Fluorescently labelled probes, which emit light (fluoresce) under certain conditions, for instance when the probe has bound to the target DNA sequence.
Identifying particular alleles of genes using DNA probes
- A DNA probe is made that has base sequences that arc complementary to pan 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 (DNA hybridisation).
- The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.
DNA hybridisation
Takes place when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases
Locating specific alleles of genes
Using DNA probes and DNA hybridisation it is possible to locate a specific allele of a gene.
Locating specific alleles of genes process
- first determine the sequence of nucleotide bases of the mutant allele we are trying to locate.
- 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 the PCR.
- A DNA probe is made by attaching a marker to the DNA fragment.
- DNA from the person suspected of having the mutant allele we want is heated to separate its two strands.
- The separated strands are cooled in a mixture containing many of our ONA probes.
- If the DNA contains the mutant allele. one of our probes is likely to bind because the probe has base sequences that are complementary to 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 which can be seen using a special microscope.
Genetic screening
- screen for mutant alleles of genetic disorders
- screen for cancer detecting oncogenes
How to gene mutations arise?
If one or more nucleotide bases in DNA are changed in any one of a variety of ways.
Benefits of genetic screening
Personalised medicine
- It allows doctors to provide advice and health care based on an individual’s genotype.
Some people’s genes can mean that a particular drug may be either more or less effective in treating a condition.
It gives insight on the dose needed, saving money that would otherwise be wasted on overprescribing the drug
Genetic counselling
Genelic counselling is like a special form of social work, where advice and information are given that enable people to make personal decisions about themselves or their offspring
- One important aspect is to research the family history of an inherited disease and to advise parents on the likelihood of it arising in their children and the emotional, psychological, medical, social and economic impacts
What is genetic fingerprinting?
A diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis.
Variable number tandem repeats (VNTRs)
DNA bases which are non-coding
- For every individual the number and length of VNTRs has a unique pattern. They are different in all individuals except identical twins
Gel electrophoresis
Used to separate DNA fragments according to their size. The DNA fragments are placed on to an agar gel and a voltage is applied across it
- The resistance of the gel means that the larger the fragments, the more slowly they move. Therefore, over a fixed period, the smaller fragments move further than the larger ones so difrerent lengths are separated.
- Their final positions 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 on the gel. Only DNA fragments up to around 500 bases long can be sequenced in this way.
- Larger genes and whole genomes must therefore be cut into smaller fragments by restriction endonucleases.
Stages of genetic fingerprinting
extraction
digestion
separation
hybridisation
development.
Genetic fingerprinting
- Extraction
The first stage is to extract the DNA by separating it from the rest of the cell. As the amount of DNA is usually small, its quantity can be increased by using the polymerase chain reaction
Genetic fingerprinting
- Digestion
The DNA is then cut into fragments, using the same restriction endonucleases. The endonucleases are chosen for their ability to cut close to, but not within, the target DNA
Genetic fingerprinting
- Separation
The fragments of DNA are next separated according to size by gel electrophoresis under the influence of an electrical voltage. The gel is then immersed in alkali in order to separate the double strands into single strands.
Genetic fingerprinting
- Hybridisation
DNA probes are now used to bind with VNTRs. The probes have base sequences which are complementary to the base sequences of the VNTRs, and bind to them under specific conditions, such as temperature and pH. The process is carried out with different probes, which bind to different target DNA sequences
Genetic fingerprinting
- Development
Finally, an X-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes. (If using fluorescent probes, the positions a re located visually.) Because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars is revealed. The pattern of the bands is unique to every individual except identical twins.
Uses of DNA fingerprinting
Genetic relationships and variability
Forensic science
Medical diagnosis
Plant and animal breeding
