Gene Mutation

 

Mutations are changes in genes, which are passed on to daughter cells. DNA is a very stable molecule, and it doesn't suddenly change without reason, but bases can change when DNA is being replicated. Normally replication is extremely accurate, and there are even error-checking procedures in place to ensure accuracy, but very occasionally mistakes do occur (such as a T-C base pair). So a mutation is a base-pairing error during DNA replication.

 

There are three kinds of gene mutation, shown in this diagram:

 

[insert image of gene mutations]

 

Substitution mutations only affect one amino acid, so tend to have less severe effects. In fact if the substitution is on the third base of a codon it may have no effect at all, because the third base often doesn't affect the amino acid coded for (e.g. all codons beginning with CC code for proline). These are called silent mutations. However, if a mutation leads to a premature stop codon the protein will be incomplete and certainly non-functional. This is called a nonsense mutation.

 

Deletion and insertion mutations have more serious effects because they are frame shift mutations i.e. they change the codon reading frame even though they don't change the actual sequence of bases. So all amino acids "downstream" of the mutation are wrong, and the protein is completely wrong and nonfunctional. However, the effect of a deletion can be cancelled out by a near-by insertion, or by two more deletions, because these will restore the reading frame. A similar argument holds for a substitution.

 

Mutation Rates and Mutagens

Mutations are normally very rare, which is why members of a species all look alike and can interbreed.

However the rate of mutations is increased by chemicals or by radiation. These are called mutagenic agents

or mutagens, and include:

• High-energy, ionising radiation such as x-rays, ultraviolet rays, a, b, or g rays from radioactive sources.

This ionises the bases so that they don't form the correct base pairs. Note that low-energy radiation

(such as visible light, microwaves and radio waves) doesn't have enough energy to affect DNA and so is harmless.

 

• Intercalating chemicals such as mustard gas (used as a weapon in warfare), which bind to DNA separating the two strands.

 

• Chemicals that react with the DNA bases such as benzene, nitrous acid, and tar in cigarette smoke.

 

• Viruses. Some viruses can change the base sequence in DNA causing genetic disease and cancer.

 

During the Earth's early history there were far more of these mutagens than there are now, so the mutation rate would have been much higher than now, leading to a greater diversity of life. Some of these mutagens are used today in research, to kill microbes or in warfare. They are often carcinogens since a common result of a mutation is cancer.

 

Mutations and Cancer

you know that mutations in some genes can cause cancer. Two of these genes are protooncogenes and tumour suppressor genes.

 

• Proto-oncogenes encode proteins that act as growth factors by stimulating cell division. The proteins allow cells to pass the checkpoints in the cell cycle and are part of the normal tight control of cell division in organisms. Normally the proto oncogene proteins are only expressed when needed for growth, but if a mutation causes a proto-oncogene to be expressed all the time, then it causes uncontrolled cell division, which can lead to cancer. The mutated gene is then called an ocogene.

 

• Tumour-suppressor genes encode proteins that inhibit cell division by blocking the cell cycle checkpoints. Some tumour suppressor genes also initiate cell death (apoptosis). Tumour suppressor genes thus override the effect of oncogenes, and so are also known as anti-oncogenes.

 

For a cancer to occur, one or more tumour suppressor genes must also be mutated, so their control is removed and cells divide out of control.

 

[image - metasasis]

 

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So a cancer is usually the result of several different mutations building up.

 

• A tumour is mass of identical cells (clones) formed by uncontrolled cell division.

 

• A benign tumour grows slowly, remains encased in a capsule and does not spread far. Benign tumours are usually harmless (e.g. warts), though they may cause harm by pressing on blood vessels or other tissues. They can often be treated easily by surgery or radiation.

 

• A malignant tumour grows quickly and spreads throughout the surrounding tissue, affecting its normal function and so causing harm (e.g. lung cancer reduces elasticity of alveoli). These tumours are more difficult to treat without damaging the whole tissue.

 

• A metastasis is a tumour that has spread to the bloodstream or lymphatic system and so can spread to other parts of the body, causing secondary tumours there. These are the most difficult to treat.

 

Stem Cells

most somatic (body) cells only express a few of their genes, so can only carry out a limited set of functions. These cells are specialised (or differentiated). 

 

In animals specialised cells are irreversibly differentiated, so they and their daughter cells will keep their specialisation and cannot become any other

kind of cell. 

 

However, there are a few cells that remain undifferentiated and so can become any type of cell. These are called stem cells.

 

Stem cells possess two key properties: -

 

• Potency – stem cells have the potential to differentiate into specialized cell types.

• Immortality – stem cells can divide indefinitely.

 

Understanding why stem cells have these properties, but all other cells do not, is a fascinating area of current research. The answers would lead to greater understanding of the process of cell differentiation and the development of cancer.

 

There are different types of stem cell:- 

 

• Totipotent (or omnipotent) stem cells can differentiate into any cell type and can even develop into a complete organism. Totipotent stem cells are found in embryos up to the 32-cell stage.

 

• Pluripotent stem cells can differentiate into nearly all cells. Pluripotent cells are found in blastocysts (i.e. five-day-old embryos containing around 150 cells, before implantation), so are called embryo stem cells.

 

• Multipotent stem cells can differentiate into a related family of cells (e.g. blood cells, muscle cells). Multipotent cells are found in some tissues throughout life, so are called adult stem cells (even though they’re present in children). They are used by the body to replace and repair damaged tissue.

 

Stem Cells in Medicine

The discovery of stem cells in the 1950s immediately led to suggestions that they could be used clinically.

 

The idea is to transplant tissue grown from stem cells into a patient, where it would grow and replace damaged tissue. Some potential examples of these cell-based therapies are shown in the table: -

 

[insert table]

 

Since the tissues could be grown from the patient’s own stem cells, there is no risk of rejection, unlike conventional transplants. Blood cell-forming (hematopoietic) stem cells from bone marrow are already being used successfully to treat leukaemia (cancer of white blood cells); and heart disease and diabetes have be treated in mice. In addition, human stem cells grown in culture are being used to test the effects of new drugs, without harming humans or animals.

​

Where do stem cells come from?

• Embryo stem cells are grown in vitro from human embryos (blastocysts) created by in vitro fertilisation. These embryos are made to help infertile couples reproduce, but any “spare” embryos, no longer needed for reproduction, can be used to create stem cells, with the informed consent of the donor couple. These stem cells are pluripotent, so can be differentiated into a wide variety of different cell types for clinical use. However, there remains a debate about whether it is ethical to use human embryos for this purpose. Embryonic stem cell research had been banned across Europe by the European parliament, although the UK government does allow such research in the UK.

 

• Adult stem cells are extracted from certain tissues of the body. It is thought that most organs andtissues contain a small number of undifferentiated stem cells, which the body uses for repair. Tissues where stem cells have been found include the brain, bone marrow, blood vessels, muscle, skin, heart, gut and liver. The use of these cells has no ethical issues, and there are also no problems of rejection if the stem cells are taken from the patient’s own body. However, they are difficult to find and difficult to grow in culture, and they are only multipotent.

 

• Induced pluripotent stem cells (iPSCs) are normal, specialised adult cells that have been genetically reprogrammed to become undifferentiated, pluripotent stem cells. iPSCs are a very new development, still at the research stage, but they may solve some of the problems of both adult and embryo stem cells.

 

Stem Cells in Plants

In plants, unlike animals, some cells remain totipotent throughout life. These cells have the ability to develop into whole plants. These totipotent plant cells are used naturally and artificially: -

 

• Vegetative Propagation. This is the natural method of asexual reproduction used by plants, where a bud grows from a vegetative (i.e. not reproductive) part of the plant (usually the stem) and develops into a complete new plant, which eventually becomes detached from the parent plant. There are numerous forms of vegetative reproduction, including: bulbs (e.g. onion, daffodil); corms (e.g. crocus, gladiolus); rhizomes (e.g. iris, couch grass); stolons (e.g. blackberry, bramble); runners (e.g. strawberry, creeping buttercup); tubers (e.g. potato, dahlia); tap roots (e.g. carrot, turnip) and tillers (e.g. grasses).

 

Many of these methods are also perenating organs, which means they contain a food store and are used for survival over winter as well as for asexual reproduction. 

 

Since vegetative reproduction relies entirely on mitosis, all offspring are clones of the parent.

 

[insert images]

 

• Cuttings. This is an old artificial method of cloning plants. Parts of a plant stem (or even leaves) are cut off and simply replanted in wet soil. Each cutting produces roots and grows into a complete new plant, so the original plant can be cloned many times. Differentiated cells from tissues in the cut stem start dividing and re differentiating to become root tissue. This division and differentiation is helped if the

cuttings are dipped in “rooting hormone” (a plant growth regulator, such as IAA). 

 

Many flowering plants, such as geraniums, African violet and chrysanthemums are reproduced commercially by cuttings.

 

[insert images]

 

• Tissue Culture (or micropropagation). This is a more modern, and very efficient, way of cloning plants. Small samples of plant tissue, called an explant, can be grown on agar plates in the laboratory in much the same way that bacteria can be grown. Any plant tissue can be used for this (e.g. from a leaf).

 

The plant tissue can be separated into individual cells, each of which can grow into a mass of undifferentiated cells called a callus. If the correct plant growth regulators (PGRs) are added these cells can develop into whole plantlets, which can eventually be planted outside, where they will grow into normal-sized plants. 

 

Conditions must be kept sterile to prevent infection by microbes.

 

[insert images]

 

With micropropagation thousands of clones of a particularly good plant can be made quickly and in a small space. Micropropagation is used on a large scale for fruit trees, ornamental plants and plantation crops such as oil palm, date palm, sugar cane and banana, that cannot be asexually propagated by other means. 

 

Because whole plants can be grown from single cells, this technique could be combined with genetic engineering to grow genetically modified plants from a single genetically-modified cell.

 

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Control of Gene Expression

 

We’ve seen how genes are expressed through transcription and translation into proteins, which give cells their functions and properties. But cells don’t express all their genes all the time. 

 

Gene expression can be switched on or off by other genes (e.g. during embryo development), by stimuli (e.g. light, injury, nutrients), or by hormones (e.g. growth hormone, oestrogen). There are five control points along the gene expression pathway:

 

[insert images]

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Control of Transcription by Transcription Factors

 

Transcription is the most important control, since it is the earliest and most efficient point, so mRNA is not made if it’s not needed. We’ve seen that, for transcription to happen, the enzyme RNA polymerase must bind to the DNA molecule just upstream of the gene, at a region called the promoter. However, RNA polymerase binds only weakly at first and it needs the assistance of a number of other DNA-binding proteins, called transcription factors, before it can start transcribing the gene. The transcription factors also bind to the promoter, upstream of the DNA polymerase. DNA is a very flexible molecule and it can loop to allow all the different transcription factors to bind together. This bonding activates RNA polymerase, which can now move along the DNA molecule, transcribing the gene.

 

[insert images]

 

This example shows two transcription factors, but in practice there can be over a dozen different proteins involved. Some promote transcription, while others inhibit it, so transcription factors provide a very flexible method of control. Since transcription factors are proteins, they are synthesised in the cytoplasm and transported into the nucleus to bind to DNA. And of course their own production is controlled by

transcription factors!

 

Steroid hormones control protein synthesis (p22), and they do this via transcription factors. For example oestrogen is secreted by the ovaries and stimulates synthesis of many proteins in target tissues such as the hypothalamus and breasts.

 

[insert images]

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1. Oestrogen, like all steroids hormones, is a lipid, so crosses the cell membrane by lipid diffusion and enters the cytoplasm.

 

2. Oestrogen binds to its receptor protein (called ERa) in the cytoplasm to form a hormone-receptor complex. This complex is now a transcription factor.

 

3. The active transcription factor diffuses into the nucleus through a nuclear pore.

 

4. In the nucleus the transcription factor binds to a specific base sequence on a DNA promoter, upstream of RNA polymerase.

 

5. This binding stimulates RNA polymerase to transcribe genes and so stimulates  protein synthesis.

 

Control of mRNA by RNA Interference

mRNA molecules have a fairly short lifetime – they are degraded by enzymes after they have been used for translation. The quicker the mRNA is broken down, the less protein can be made, and this is another point where gene expression can be controlled. This control is carried out by special RNA molecules called small interfering RNA (siRNA) and the process is called RNA interference (RNAi).

 

1. siRNA is a short double-stranded RNA molecule, about 20 base pairs long. It is made by special regulatory genes. These genes are transcribed as normal to make single-stranded RNA, which then folds back on itself by complementary base pairing to make a hairpin-like double-stranded molecule.

 

2. In the cytoplasm siRNA binds to a protein called the RNA-induced silencing complex (RISC).

 

3. RISC breaks the double-stranded siRNA into its separate strands. One strand remains attached to the RISC protein, while the other strand is discarded.

 

[insert images]

 

4. The RISC-RNA complex now binds to mRNA molecules in the cytoplasm by complementary base pairing. Any mRNA molecules with a base sequence complementary to the 20-base siRNA sequence will bind.

5. This binding causes RISC to cut the mRNA molecule in two.

6. This cleaved mRNA can no longer be used in translation, and is broken down by nuclease enzymes.

​

RNA interference seems to be mainly used by cells in defence against viral attack. Viruses enter a host cell and use the host cell’s ribosomes to translate viral mRNA and so make more viral proteins. In defence, host cells make siRNA with complementary sequences to viral mRNA, so preventing synthesis of viral proteins.

 

Applications of RNA interference

 

Since the discovery of RNA interference in 1998, scientists quickly realised that they could use siRNA to “silence” genes. A 20-base pair siRNA molecule can be synthesised in the laboratory with a sequence that is complementary to part of the gene that is to be silenced. This siRNA is then introduced into cells to observe the effect of silencing that gene. 

 

Applications include: -

 

• Identifying the function of genes in cells or simple organisms by silencing the gene and observing the functional effects.

 

• Genetically-modifying crop plants by silencing undesirable genes e.g. those that make toxins or allergens. In the Flavr Savr tomato a gene causing ripening was silenced, causing the tomatoes to stay fresh for longer.

• ​

• Treating human patients to silence essential genes in cancer cells, or in antiviral therapies e.g. silencing the gene for the receptor protein used by HIV.

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Biotechnology

 

Biotechnology usually refers to the application of molecular (DNA) biology in the laboratory. 

 

Biotechnology has applications in: -

 

• research e.g. human genome project

• medicine e.g. genetically-engineered drugs, gene therapy

• agriculture e.g. improving crops

• industry e.g. manufacturing enzymes, biosensors

 

A particular aspect of biotechnology is genetic engineering, which means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Genetic engineering can include inserting a foreign gene from one species into another (forming a transgenic organism); altering an existing gene so that its product is changed or changing gene expression.

 

Techniques of Biotechnology

Modern biotechnology is possible due to the development of techniques from the 1960s onwards, which arose from our greater understanding of DNA and how it functions, following the discovery of its structure by Watson and Crick in 1953. This table lists the techniques that we shall look at in detail.

 

[insert table of biotech]

 

1. Restriction Enzymes

 

These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut phosphodiester bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends.

 

[insert images]

 

The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another sticky end by complementary base pairing (i.e. with weak hydrogen bonds), but only if the sticky ends have both been cut with the same restriction enzyme so that they have complementary sequences. Restriction enzymes have highly specific active sites, and will only cut DNA at specific base sequences, 4-8 base pairs long, called recognition sequences. Recognition sequences are usually palindromic, which means that the sequence and its complement are the same but reversed (e.g. GAATTC has the complement CTTAAG).

 

 

Restriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict” viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences. Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R, and HindIII is from Haemophilis influenzae.

 

2. DNA Ligase

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. Ligase is therefore a bit like DNA polymerase. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments.

 

Two restriction fragments can anneal if they have complementary sticky ends, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. DNA ligase completes the DNA backbone by forming covalent phosphodiester bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.

 

[insert images]

 

3. Reverse Transcriptase

The enzyme reverse transcriptase does the reverse of transcription: it synthesises DNA from an RNA template (so it is an RNA-dependent DNA polymerase enzyme). 

 

Reverse transcriptase is produced naturally by a group of viruses called the retroviruses (which include HIV), and it helps them to invade cells.

 

In biotechnology reverse transcriptase is used to make an “artificial gene”, called complementary DNA (cDNA), from an mRNA template as shown in this diagram:

 

[insert images]

 

Mature mRNA (without introns) is extracted from cells and mixed with reverse transcriptase and DNA nucleotides. A new strand of DNA is synthesised, complementary to the mRNA strand, forming a doublestranded DNA/RNA “heteroduplex” molecule. The two strands of this molecule are then separated and reverse transcriptase now synthesises a second DNA stand, complementary to the first. The result is a normal double-stranded DNA molecule called cDNA. Note that the cDNA molecule is much shorter than the original gene in the organism’s DNA (typically <50% the size), since the cDNA doesn’t have introns. cDNA is therefore an “artificial gene”.

 

Reverse transcriptase has several uses in biotechnology:

 

• It makes genes without introns. Eukaryotic genes with many introns are often too big to be incorporated into a bacterial plasmid, and bacteria are unable to splice out the introns anyway. The artificial cDNA gene is made from mRNA that already has the introns spliced out of it, so it can be expressed in bacteria.

 

• It makes a stable copy of a gene, since DNA is less readily broken down by enzymes than RNA.

 

• It makes genes easier to find. There are some 20 000 genes in the human genome, and finding the DNA fragment containing one gene out of this many is a very difficult task. However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule. For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.

 

4. Electrophoresis [link to video]

This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively charged phosphate group, so DNA is attracted to the anode (the positive electrode). 

 

The molecules have to diffuse through the gel, and longer lengths of DNA are retarded by the gel so move more slowly than shorter lengths. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.

 

[insert images]

 

Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are two common

methods for doing this:

 

• The DNA can be stained with a coloured chemical such as azure A (which stains the DNA bands blue), or a fluorescent molecule such as ethidium bromide (which emits coloured light when the finished gel is illuminated with invisible ultraviolet light).

 

• The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32P, then visualised using autoradiography. Ordinary photographic film (sometimes called X-ray film) is placed on top of the finished gel in the dark for a few hours, and the radiation from any radioactive DNA on the gel exposes the film. When the film is developed the position of the DNA shows up as dark bands on the film. This method is extremely sensitive.

 

[insert images]

 

5. DNA Sequencing

 

This means reading the base sequence of a length of DNA. DNA sequencing is based on a beautifully elegant technique developed by Fred Sanger in Cambridge in 1975, and now called Sanger Sequencing.

 

1. Label 4 test tubes A, T, C and G. Into each test tube add: a sample of the DNA to be sequenced (containing many millions of individual molecules); a radioactive primer (so the DNA can be visualised later on the gel); the four DNA nucleotides and the enzyme DNA polymerase.

 

1. In each test tube add a small amount of a special modified dideoxy nucleotide that cannot form a phosphodiester bond and so stops further synthesis of DNA. Tube A has dideoxy A (A*), tube T has dideoxy T (T*), tube C has dideoxy C (C*) and tube G has dideoxy G (G*). The dideoxy nucleotides are present at about 1% of the concentration of the normal nucleotides.

 

1. Let the DNA polymerase synthesise many copies of the DNA sample. From time to time at random a dideoxy nucleotide will be added to the growing chain and synthesis of that chain will then stop. A range of DNA molecules will be synthesised ranging from full length to very short. The important point is that in tube A, all the fragments will stop at an A nucleotide. In tube T, all the fragments will stop at a T nucleotide, and so on.

 

1. The contents of the four tubes are now run side by side on an electrophoresis gel, and the DNA bands are visualised by autoradiography. Since the fragments are now sorted by length the sequence can simply be read off the gel starting with the smallest fragment (just one nucleotide) at the bottom and reading upwards.

 

[insert images]

 

There is now a modified version of the Sanger method called cycle sequencing, which can be completely automated. The primers are not radiolabelled, but instead the four dideoxy nucleotides are fluorescently labelled, each with a different colour (A* is green, T* is red, C* is blue and G* is yellow). The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the process. The resulting mixture is separated using capillary electrophoresis, which gives good separation in a single narrow tube gel. The gel is read by a laser beam and the sequence of colours is converted to a DNA sequence by computer program (like the screenshot below). This technique can sequence an amazing 12 000 bases per minute.

 

Many thousands of genes have been sequenced using these methods and the entire genomes of several

organisms have also been sequenced. A huge project to sequence the complete 3-billion base sequence of the human genome was recently completed. This information is giving us unprecedented knowledge about ourselves, and is likely to lead to dramatic medical and scientific advances. Once a gene sequence is known the amino acid sequence of the protein that the DNA codes for can also be determined, using the genetic code table. 

 

The sequence can also be compared with DNA sequences from other individuals and even other species to work out relationships between individuals or species.

 

These genome sequences represent vast amount of data that must be analysed and compared to existing sequences. Powerful computers, huge databases and intelligent search programs are being developed to deal with this data, which has led to a whole new branch of biology called bioinformatics, and a new way of doing biology without touching a living thing: in silico.

 

6. Restriction Mapping

 

A restriction map is simply a diagram of a piece of DNA marked with the locations of sites where it is cut by restriction enzymes.

 

A piece of DNA is cut with two different restriction enzymes, both on their own, and together. This gives three different mixtures of restriction fragments, which are run on an electrophoresis gel (labelled E1, E2 and E1+E2 on the gel below). The first lane on this gel contains a “DNA ladder” – a mixture of DNA fragments of known sizes – which is used to calibrate the gel. By comparison with the ladder bands, the length of each restriction fragment can be measured (marked in kilobases, kb on this diagram). 

 

[insert images]

 

From this information alone we have to deduce the restriction map. Firstly, the fragment lengths in each lane add up to 17, so we know the original DNA was 17kb  long. There must be two recognition sites for restriction enzyme 1 (E1), since it gave three bands, while there must be just one recognition site for restriction enzyme 2 (E2), since it gave two bands. By a process of logic, we can construct the following restriction map to account for the banding pattern.

 

[insert images]

 

This map is useful in itself, as it can be used to choose restriction enzymes to generate known-sized fragments. But it is even more useful as an aid to DNA sequencing. A long piece of DNA can be cut into shorter restriction fragments, which are easy to sequence. A computer then uses the restriction map to “stitch together” the individual sequences in the correct order and orientation, to generate the complete sequence. The human genome was sequenced in this way.

 

7. Polymerase Chain Reaction (PCR) [link to video]

The polymerase chain reaction is a technique used to copy (or amplify) DNA samples as small as a single molecule. It was developed in 1985 by Kary Mullis, for which discovery he won a Nobel Prize in 1993. PCR is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times. 

 

The details are shown in this diagram:

 

[insert images] [video]

 

1. Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase.

 

2. Heat to 95°C for two minutes to breaks the hydrogen bonds between the base pairs and separate the two strands of DNA. Normally (in vivo) the DNA double helix would be separated by an enzyme.

 

3. Add primers to the mixture and cool to 40°C. Primers are short lengths of single stranded DNA (about 20 bp long) that anneal (i.e. form complementary base pairs) to complementary sequences on the two DNA strands forming short lengths of double-stranded DNA. The DNA is cooled to 40°C to allow the hydrogen bonds to form. There are two reasons for making short lengths of double-stranded DNA:

• The enzyme DNA polymerase requires some existing double stranded DNA to get it started.

• Only the DNA between the primer sequences is replicated, so by choosing appropriate primers you can ensure that only a specific target sequence is copied.

 

4. The DNA polymerase enzyme can now build new stands alongside each old strand to make doublestranded DNA. Each new nucleotide binds to the old strand by complementary base pairing and is joined to the growing chain by a phosphodiester bond. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible.

 

5. Each original DNA molecule has now been replicated to form two molecules. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. After n cycles, there is an amplification factor of 2n. Typically PCR is run for 20-30 cycles.

 

[insert images/video]

 

PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning, electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that’s been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases.

 

8. Southern Blot

A Southern blot is used to detect a specific target sequence in samples of DNA. DNA samples separated in an electrophoresis gel can’t be manipulated or stored, since the electrophoresis gel is fragile, and the separated DNA fragments continue to diffuse through it, blurring the bands. So, in a blot, the DNA is transferred to a stronger substrate, where it can be fixed. The target sequence is then detected using a DNA probe.

 

A DNA probe is simply a short length of single-stranded DNA (100-1000 nucleotides long) with a label attached. There are two common types of label used:

 

A radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using

autoradiography.

 

A fluorescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light.

Probes can be made to fluoresce with different colours.

 

If a probe is added to a mixture of different pieces of single-stranded DNA (e.g. restriction fragments) it will anneal to any fragments of DNA containing the complementary sequence forming regions of doublestranded DNA. This double-stranded DNA is a mixture or hybrid (since it contains DNA from two different sources), so the DNA formed is called hybrid DNA, and the process is called hybridisation. The hybrid DNA fragments will now be labelled and will stand out from the rest of the DNA.

 

The Southern blot method: -

1. DNA is extracted from the source cells (e.g. different patients or different species) and amplified by PCR to make enough DNA for the hybridisation. The DNA samples are then digested by a restriction enzyme into many small fragments, and the fragments separated on an electrophoresis gel.

 

1. The gel is placed in an alkali solution, which breaks the hydrogen bonds between the DNA bases causing the stands to separate.

 

1. A thin sheet of nylon or nitrocellulose is placed on top of the gel and covered with a stack of paper towels, weighted down to make good contact. The alkali solution is drawn up through the gel to the paper towels by capillary action, bringing the DNA with it. The negatively-charged DNA sticks to the positively-charged nylon membrane.

 

1. After a few hours the DNA fragments are all transferred to the nylon sheet (though of course they can’t be seen). The nylon sheet is separated from the gel, and treated with UV light to fix the DNA molecules.

 

1. The nylon sheet is placed in a plastic bag containing a solution of labelled probes and mixed thoroughly. The probes will anneal by complementary base-pairing to DNA fragments in the nylon membrane that have a complementary sequence, forming hybrid DNA molecules stuck to the nylon sheet (but again they can’t be seen). 

 

1. The location of the hybrid DNA can be visualised by different methods, depending on the label used. If the probes were radioactive then they can be visualised by autoradiography, and the probes show up as bands on photographic film. If the probes were fluorescent then they can be visualised as bands of light when illuminated by ultraviolet light.

 

The Southern blot has many uses:

 

• To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from an entire genome

 

• To identify genes in one species that are similar to those of another species.Genes are remarkably similar in sequence from one species to another, so for example a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided the identification of human genes.

 

• To identify the short DNA sequences used in DNA fingerprinting.

 

• To screen for genetic diseases, such as Huntington’s disease and cystic fibrosis.

 

• The Southern blot was invented by Edwin Southern at Edinburgh University in 1975 (as a biological joke, similar blotting techniques using RNA or protein are called northern and western blots respectively).

​

9. Genetic Fingerprinting [links to video]

Genetic fingerprinting is used to distinguish between DNA samples from different people. Although 99.9% of human DNA is the same in every person, there is enough variation in the remaining 0.1% (over 100 000 base pairs) to be used to distinguish one individual from another. The differences are found in non-coding

 

DNA since these regions can accumulate mutations without affecting function, and the differences are largely due to the presence of repetitive sequences. There are different kinds of repetitive sequences: -

 

Satellite DNA – any DNA region with repetitive sequences. Satellite DNA gets its name because it forms a separate (or satellite) band when separated by density centrifugation.

 

Tandem repeat – when a simple sequence of bases in a DNA molecule is repeated without a break e.g. ATATATAT (a 2-base repeat) or CCTAAGCCTAAGCCTAAG (a 6-base repeat).

 

STR (Short Tandem Repeat) or Microsatellite – a tandem repeat with a sequence of less than10 bases.

 

Minisatellite – a tandem repeat with a sequence of 10–60 bases.

 

VNTR (Variable Number Tandem Repeat) – a region of DNA containing a particular tandem repeat. While everyone has the same repeating sequence in this region, different individuals have different numbers of repeats. [link to video]

 

Polymorphism – a range of different phenotypes of a characteristic or genotypes of a gene within a population. In molecular biology it means a range of different DNA sequences at a particular locus.

 

RFLP (Restriction Fragment Length Polymorphism, pronounced “riff-lip”) – when restriction fragments of the same region of DNA have different lengths in different people.

 

Genetic Fingerprinting was invented by Sir Alec Jeffreys at Leicester University in 1984. Today there are many different methods of genetic fingerprinting, but Jeffreys’ original method was based on Restriction Fragment Length Polymorphism (RFLPs): -

 

1. Samples of DNA to be compared (from different individuals) are amplified by PCR. The PCR primers are designed to target and copy a region of the human DNA containing VNTRs. 

 

1. The amplified DNA of the VNTR region is digested by a restriction enzyme. The same recognition sequences should be present in the different samples, so the same number of fragments should be produced, but the fragments will be different lengths depending on the number of tandem repeats in each restriction fragments.

 

1. The digested DNA is run on an electrophoresis gel to separate out the fragments by size, and the fragments are visualised using a Southern blot. The pattern of bands is different for the two samples because of the restriction fragment length polymorphism i.e. the “same” restriction fragments have different lengths due to different numbers of tandem repeats.

 

Samples of the same DNA (or from identical twins) give the same banding pattern, but samples from different people give different banding patterns.

 

[insert images]

 

This original fingerprinting method takes a few days to carry out and can produce results that are difficult to interpret. Newer methods of genetic fingerprinting have been developed that offer improved speed, sensitivity, discrimination and reliability, but the same principles are used. In the current technique, called DNA profiling and used by the police in most countries of the world, 10-13 different regions containing variable numbers of short tandem repeats (STRs) are targeted and amplified by PCR and then sorted and sized using an automated capillary sequencer. This avoids the need for restriction enzymes or Southern blot and means the process can be automated. The size data can also be stored on computers, forming National DNA Databases. It is always possible that two unrelated people might by chance have the same genetic fingerprint, but using DNA profiling it is calculated that the chance of this is less than 1 in 109.

 

DNA fingerprinting is used

• in forensic science, to match DNA samples collected from a crime (e.g. from sperm, blood, hair, skin) with that of suspects. Normally suspects are arrested because of some other link to the crime, but with the development of a national DNA database, that doesn’t have to be the case. Convictions based on DNA evidence can be difficult due to problems of contamination and poor laboratory technique.

 

• to determine family relationships, usually between a child and a suspect father (paternity testing). Since children inherit half their DNA from each parent, they inherit half their DNA fingerprint fragments from each parent, so 50% of the bands should match their mother’s bands and 50% match their father’s bands.

 

• to prevent undesirable inbreeding during breeding programs in farms and zoos. It can also identify plants or animals that carry a particular desirable (or undesirable) allele.

 

• to determine relationships between ancient humans (using DNA extracted from archaeological remains) and modern humans.

 

• to establish evolutionary relationships between different species.

 

• to measure genetic diversity within a population.

 

 

10. Vectors

Now we turn to genetically-modifying living cells. The next three techniques are concerned with transferring genes (DNA) from the test tube into cells, so that the genes can be replicated and expressed in those cells. To do this we first need a vector.

 

In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties:

 

• It is big enough to hold the gene we want (plus a few others), but not too big.

 

• It is circular (or more accurately a closed loop), so that it is less likely to be broken down (particularly in prokaryotic cells where DNA is always circular).

 

• It contains control sequences, such as a replication origin and a transcription promoter, so that the gene will be replicated, expressed, or incorporated into the cell’s normal genome.

 

• It contains marker genes, so that cells containing the vector can be identified.

 

Plasmids are the most common kind of vector, so we shall look at how they are used in some detail.

 

Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells, so bacterial cells will readily take up a plasmid. 

 

Because they are so small, plasmids are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

 

One of the most common plasmids used is the R-plasmid. This plasmid contains a replication origin, several recognition sequences for different restriction enzymes (with names like PstI and EcoRI), and two marker genes, which in this case confer resistance to antibiotics. The R plasmid gets its name from these resistance genes.

 

The diagram below shows how a gene can be incorporated into a plasmid using restriction and ligase

enzymes.

 

[insert images]

 

1. A restriction enzyme (Pst1 here) is used to cut the gene from the donor DNA, with sticky ends.

 

2. The same restriction enzyme cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later).

 

3. The gene and plasmid are mixed in a test tube and they anneal because they were cut with the same restriction enzyme and have the same sticky ends.

 

4. The fragments are joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA).

 

5. Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. These different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid vector.

 

Transformation

 

Transformation means inserting new DNA (usually a plasmid) into a living cell (called a host cell), which is thus genetically modified, or transformed. A transformed cell can replicate and express the genes in the new DNA. DNA is a large molecule that does not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell.

 

• Heat Shock. Bacterial and animal cells in culture can be made to take up DNA from their surroundings by raising the temperature suddenly raised by about 40°C.

 

• Electroporation. The most efficient method of delivering genes to bacterial cells is to use a high voltage pulse, which temporarily disrupts the membrane and allows the plasmid to enter the cell.

 

• Micro-Injection. To transform individual cells, such as fertilised animal egg cells, the DNA is injected directly into the nucleus using an incredibly fine micro-pipette.

 

• Gene Gun. Tiny gold particles coated with DNA can be fired at plant cells using a compressed air gun. The particles can penetrate the tough cell wall and deliver the DNA to the nucleus.

 

• Plant Tumours. Plant cells are infected with a transformed bacterium, which inserts its plasmid into the plant cells' chromosomal DNA. Whole new plants are grown from these cells by micropropagation.

 

• Liposomes. Human cells in vivo can be transformed by DNA encased in liposomes, which fuse with the cell membrane, delivering the DNA into the cell.

 

• Viruses. Human cells in vivo can be infected by genetically-engineered viruses, which deliver the DNA into host cells. The viruses must first be made it safe, so they can’t cause disease.

 

11. Marker Genes

Marker genes (or reporter genes) are used to find which cells have actually taken up the hybrid vector. Following transformation, there are (at least) these four possible outcomes: -

 

[insert images]

 

Vectors contain two different marker genes, which are needed to identify the required cells.

 

The first marker gene distinguishes between cells that have taken up a plasmid from those that haven’t. The plasmid contains a gene for resistance to an antibiotic such as tetracycline, so bacterial cells taking up this plasmid can make this gene product and so are resistant to this antibiotic. The cells are therefore grown on a medium containing tetracycline, which kills all the normal untransformed cells (>99.99%). 

 

Only the few transformed cells will survive, and these can then be grown and cloned on another plate.

 

The second marker gene distinguishes between cells that have taken up the hybrid plasmid from those that have taken up the original plasmid. The trick here is that the foreign DNA is inserted inside the second marker gene, so cells with the hybrid plasmid cannot make that gene product. Different genes are used for this second marker: -

 

• The marker gene can be a gene for resistance to another antibiotic, such as ampicillin. In this case, cells with the hybrid vector are not resistant to ampicillin. 

 

Since this means killing the cells we want, the ampicillin test is done on a replica plate, where the colonies are transferred to a second agar plate using a cloth stamp. Colonies that grow on the first (tetracycline) plate but not on the replica (ampicillin) plate are the ones we want.

 

 

• The marker gene can be a gene for the enzyme b-galactosidase (lactase). This enzyme turns a white substrate in the agar plate into a blue product. So colonies of cells with the original plasmid turn blue, while those with the hybrid plasmid remain white, and can easily be identified.

 

• The marker gene can be a gene for green fluorescent protein (GFP). Colonies of cells with the original plasmid fluoresce green in UV light, while those with the hybrid plasmid do not fluoresce, and can easily be identified.

 

Gene Cloning

Gene cloning simply means make multiple copies of a piece of DNA. It is a necessary step in just about any aspect of molecular biotechnology, such as genetic engineering, genome sequencing or genetic fingerprinting. There are two different ways to clone DNA: -

 

• In vitro gene cloning uses PCR to clone DNA in the test tube.

• In vivo gene cloning uses restriction enzymes, vectors, DNA ligase, transformation of bacterial cells, marker genes and growth cultures to clone DNA inside bacterial cells.

 

The two techniques have different advantages and disadvantages: -

 

[insert table comparing in vitro cloning (using PCR) V in vivo cloning (using living cells) ]

 

Genetically Modified Organisms

 

We have looked at some of the many techniques used in biotechnology. We’ll now turn to some applications of these techniques. The applications involve altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. If a foreign gene in copied from one species into another the GMO is called a transgenic organism, but remember that not all GMOs are transgenic: the genetic modification might just alter an existing gene so that its product is changed or change its gene expression. We’ll consider the applications in three groups.

 

• Gene Products Using genetically modified organisms (usually microbes) to produce chemicals (usually proteins) for medical or industrial applications.

 

• New Phenotypes Using gene technology to alter the characteristics of organisms (usually farm animals or crops).

 

• Gene Therapy Using gene technology on humans to treat a disease.

Gene Products.The biggest and most successful kind of genetic engineering is the production of gene products. These products are of medical, agricultural or commercial value to humans. This table shows a few of the examples of genetically engineered products that are already available.

 

[insert table]

 

The products are mostly proteins, which are produced directly when a gene is expressed, but they can also be non-protein products produced by genetically engineered enzymes. The basic idea is to transfer a gene (often human) to another host organism (usually a microbe) so that it will make the gene product quickly, cheaply and ethically. It is also possible to make “designer proteins” by altering gene sequences, but while this is a useful research tool, there are no commercial applications yet.

 

Since the end-product is just a chemical, in principle any kind of organism could be used to produce it. By far the most common group of host organisms used to make gene products are the bacteria, since they can be grown quickly and the product can be purified from their cells. Unfortunately bacteria cannot always make human proteins, and so fungi, animals and plants have also been used to make gene products. This table shows some of the advantages and disadvantages of using different organisms for the production of genetically-engineered gene products.

 

[insert table Ad V DisAd]

 

 

New Phenotypes

This means altering the characteristics of organisms by genetic engineering. The organisms are generally commercially-important crops or farm animals, and the object is to improve their quality in some way. This can be seen as a high-tech version of selective breeding, which has been used by humans to alter and improve their crops and animals for at least 10 000 years. This table gives an idea of what is being done.

 

[insert table - organisms and their modifications]