DNA Probes, Electrophoresis and PCR

Locating a desired DNA sequence

A DNA probe is a short single-stranded section of DNA that is complementary to the section of DNA being investigated. The probe is labelled in one of two ways:

1. Using a radioactive marker so that the location can be revealed by exposure to photographic film
2. Using a fluorescent marker that emits a colour on exposure to UV light
Copies of the probe are added to a sample of DNA fragments and will anneal to any fragment where a complementary base strand is present:
DNA probe
Isolating a desired DNA sequence
  1. DNA samples are treated with restriction enzymes to cut them into fragments
  2. The DNA samples are placed into wells cut into the negative electrode end of an agarose gel
  3. A DNA standard is added to one well – the fragments are of known length so can be used to estimate the size of the fragments in the samples
  4. The gel is immersed in a tank of buffer solution and an electric current is passed through the solution for around 2 hours
  5. DNA is negatively charged because of its phosphate groups so is attracted to the positive electrode
  6. Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run
  7. The position of the fragments can be shown by using a dye that stains DNA molecules (see above)

electrophoresis

You could try carrrying out this procedure in a virtual lab by clicking here!

Amplifying a DNA sequence

PCR stands for the polymerase chain reaction. It is used to make lots of copies of DNA and is even used by the police to give a large enough quantity of DNA for testing from the smallest blood or other sample.

  1. The DNA sample is mixed with a supply of DNA nucleotides and DNA polymerase
  2. The mixture is heated to 95°C. This breaks the hydrogen bonds holding the strands together, so the samples are now single stranded
  3. Primers (short lengths of single stranded DNA) are added
  4. The temperature is reduced to 55°C to allow the primers to bind and form small sections of double stranded sections;
  5. DNA polymerase can bind to these double-stranded sections;
  6. The temperature is raised to 72°C. The enzyme extends the double stranded section by adding nucleotides to the unwound DNA;
  7. When the DNA polymerase reaches the other end of the DNA, a new, double stranded DNA molecule is generated;
  8. The whole process can be repeated many times so the amount of DNA increases exponentially.

PCR

You can watch this procedure being carried out by clicking here and going to ‘techniques – amplifying’ or you could carry out the procedure for yourself in a virtual lab here!

Cloning In Plants And Animals

identical_triplets

Clones are abundant in nature – when a zygote splits in two identical twins are formed, when bacteria divideasexually by binary fission the resulting bacteria are clones of the original bacterium, when plants reproduce asexually by producing runners the resulting plants are clones and when a singles cell divides during mitosis the two daughter cells are clones. These clones produced by asecual reproduction are advantageous as the process is quick, can be carried out when sexual reproduction cannot and all of the offspring have the genetic information to allow them to survive in their environment. However, asexual reprodcution generates no genetic variation, unless a mutation occurs, and so all of the offspring are equally suscebtible to envorinomental changes such as the introduction of a new disease-causing pathogen.

There are two main types of cloning – reproductive and non-reproductive. Reproductive cloning is the production of offspring which are genetically identical to either their mother, if created by nuclear transfer, or the other offspring, if created by splitting embryos. Non-reproductive cloning is the use of stem cells in order to generate replacement cells, tissues or organs which may be used to treat particular diseases or conditions in humans.

Splitting embryos is the process of seperating out cells from a developing embryo and so producing separate, genetically identical organisms:

splitting embryos

Nuclear transfer is when the nucleus of a differentiated adult cell is placed in an enucleated egg cell:

nuclear transfer

Advantages of artficially cloning aniamls:

  • High value animals, such as cows with a high milk yield, can be cloned in large numbers.
  • Rare animals can be cloned in order to preserve the species.
  • Genetically modified animals, such as goats which produce spider silk in their milk, can be quickly produced.

Disadvantages of artifically cloning animals:

  • As with asexual reproduction, the genetic unformity of cloning means that all of the offspring will be susceptible to environmental changes.
  • Animal welfare is not always taken into account, for example chickens with a high meat yield, yet are unable to walk, have been produced.
  • It is not yet known if animals cloned using nuclear material from adult cells will develop any long term health problems.

An example of natural vegetative propogation is the English Elm tree. After damage to the parent plant, such as disease or burning, root suckers (basal sprouts) begin to grow from the meristem tissue in the trunk close to the ground, as this is where the lest damage is likely to have occured. This response to stress or trunk death helps the elm to spread, for example, when an elm is felled during copicing, the root suckers grow into a circle of new elms, called a clonal patch, around the old trunk. These new elms produce their own root suckers, and so the clonal patch continues to expand where resources permit it. However, this adaptation can also be disadvantageous, in particular when it is in response to Dutch elm disease. The roots of an elm infected with dutch elm disease will produce many root suckers, but as these suckers are clones of the original plant they have no resistance to the fungal attack and so as they continue to grow they also begin to show symptoms of the disease. Many plants we take for granted also use vegetative propogation as a survival mechanism – potatoes form tubers which are underground stems swollen with nutrients from which new plants grow, onions and daffodils form bulbs which are condensed shoots containing nutrients and from which new bulbs can fom and strawberries have specialised stems, called runners (see below), which grow along the ground, forming new roots and shoots at the tips. These adaptations all mean the plant can reproduce even if it becomes isolated and there is no reliance on wind, insects or other pollinating agents, but the tubers and bulbs are also a disadvantage as they are an attractive foood source for certain animal including us humans.

natural_vegetative_propogation

Plants can also be propogated aritifically. Traditionally there were two methods of doing this; taking cuttings and grafting:

Taking cuttings is where a stem is cut between nodes and its lower leaves are removed. The cut end is then treated with plant hormones to encourage root growth  before planting. The cuttingss are clones of the parent plant. Commercially this techniques is used to quickly produce large numbers of plants such as geraniums.

cuttings

Grafting is where a shoot section of a woody plant, such as a rosebush or fruit tree, is joined to a rootstock (a root and stem already growing). The graft then grows and is a clone of the original plant, but the rootstock is genetically different.

grafting

Although these methods are useful they cannot easily produce high numbers of plants and some plants struggle to reproduce successfully in these ways. The more mordern method of artificial vegetative propogation is micropropogation by callus tissue culture. This method can quickly produce very large stocks of a plant from a small amount of plant tissue, and it has an added advantage that the stock is disease free. Many household plant, such as orchids, are produced using the following method:

  1. A small piece of tissue (an explant) is taken from the shoot tip of a plant.
  2. The explant is placed on a nutrient growth medium and cells in the tissue divide to form a callus (a mass of undifferentiated cells).
  3. Single callus cells are seperated from the mass and placed on a growing medium with plant hormones that encourage shoot growth.
  4. These growing shoots are then transferred to another medium with hormones encouraging root growth.
  5. Growing plants are then transferred to a greenhouse to acclimatise and grow further, before being planted outside.

micropropogation

Plant cloning in agriculture has both advantages and disadvantages. Lots of genetically identical plants can be produced from one plant – you know what the plants will be like and the process is faster than selective breeding. Also, costs are reduced as the crop is all ready at the same time and plants can be produced at any time of the year instead of having to wait until their natural growing season. However, the process is arguably more labour intensive as it’s harder to replant small plants than sow seed. Most importantly, environmental change such as the arrival of a new disease could damage the whole crop as their identical genetics means that they would all be equally susceptible.

Meiosis

Meiosis 1

Prophase 1:

prophase1

  • Chromatin condenses and supercoils so chromosomes shorten and thicken.
  • Chromosomes come together in their homologous pairs to form a bivalent.
  • The non-sister chromatids wrap around each other and attach at chiasmata. Sections may be swapped in crossing over.
  • Nucleolus disappears and nuclear envelope disintegrates.
  • A spindle (made of microtubules) forms.

Metaphase 1

  • Bivalents line up across equator of spindle, attached to spindle fibres at the centromeres.
  • They are arranged randomly with each member of a homologous pair facing opposite poles.

Anaphase 1

anaphase1

  • The homologous chromosomes in each bivalent are pulled by the spindle fibres to opposite poles. The centromeres do not divide. The chiasmata separate (and swapped sections stay swapped).

Telophase 1

telophase1

  • In animals two new nuclear envelopes form and the cell divides by cytokinesis.

Meiosis 2 (division is in a plane at right angles to meiosis 1)

Prophase 2:

  • Reformed nuclear envelopes break down, the nucleolus disappears, chromosomes condense and spindles form.

Metaphase 2:

  • Chromosomes line up on equator of spindle and are attached to the spindle fibres at centromeres.
  • The chromatids of each chromosome are randomly assorted.

Anaphase 2:

  • The centromeres dvide and the chromatids are pulled to opposite poles by the spindle fibres. The chromatids randomly segregate.

Telophase 2

  • Nuclear envelopes reform around the haploid daughter nuclei
  • In animals the two cells now divide to give four haploid cells
  • In plants a tetrad of four haploid cells is formed.

Tourism!

With the January exams looming I thought I’d share my revision notes for the geography unit on tourism 🙂

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Diet and Food Production

Balanced Diet: a diet which contains all the nutrients required for health in the appropriate proportions.

Balanced

  • Obesity is when a person is 20% or heavier than the recommended weight for their height. Obesity is caused through malnutrition, which is where a person’s diet is unbalanced. In this case of malnutrition, the person consumes too much energy and the excess is deposited as fat in the adipose tissues which can impair health. The conditions most commonly associated with obesity are cancer, cardiovascular disease, type 2 diabetes, gallstones, osteoarthritis and high blood pressure (hypertension).

HDL and LDL

  • High blood cholesterol is linked with coronary heart disease. Although it’s found in cell membranes, the skin and is used to make steroid sex hormones and bile, above 5.2mmol/dm3 is harmful. Cholesterol is insoluble but is carried by HDL’S and LDL’s. Lipoproteins are a combination of lipid, cholesterol and protein.
    •  HDL’s contain unsaturated fat and tend to carry cholesterol from the body tissues back to the liver where it’s used in cell metabolism to make bile or is broken down, meaning high levels of HDL’s are associated with reducing blood cholesterol levels. They reduce deposition of cholesterol in the artery walls (called atherosclerosis) and may help to remove the depositions already there.
    • LDL’s contain saturated fats. They tend to carry cholesterol to body tissue from the liver. The concentration of LDL’s in the blood increases when a diet contains too much saturated fat and cholesterol. High LDL levels result in deposition in the artery walls.
      • Saturated fats decrease the activity of LDL receptors in body tissue so as the concentration of blood LDL’s rises; less is removed resulting in higher LDL concentrations and more deposition. Polyunsaturated and monounsaturated fats increase the activity of LDL receptors so more is removed from the blood.
    • Coronary heart disease: a condition in which the coronary arteries narrow from an accumulation of plaque (atherosclerosis) and cause a decrease in blood flow.
  • Humans depend on plants for all food as they are the basis of all food chains. This is because humans eat both plants directly, and herbivores who eat plants.
  • Making food production more efficient:
    • Plants:
      • Improve growth rate, increase yield, reduce crop loss due to diseases/pests, easier harvest by standardising crop size, and improve response to fertilisers.
    • Animals:
      • Improve growth rate, increase productivity, and increase resistance to disease
    • Selective breeding:
      • Isolation → artificial selection → inbreeding/line breeding
      • A pair of animals displaying desirable characteristics is allowed to reproduce. The offspring are sorted and those with the best combinations of characteristics are allowed to reproduce. Over many generations this exaggerated the desired characteristic.
      • Selecting which animals can breed or which seeds to be sown is known as applying selection pressure.
    • Pesticides, fungicides and fertilisers increase plant yield as they are less likely to die from insects eating them or a fungal disease, and the fertilisers provide all the minerals the plant needs to grow so it can grow faster. Antibiotics can increase food production of animals as fewer animals die from disease so a higher yield is achieved.

bacteria2

  • Methods of preventing food spoilage;
    • Cooking – denatures enzymes and other proteins killing microorganisms
    • Pasteurising – heat then rapid cooling kills microorganisms
    • Drying/salting/coating in sugar all dehydrate microorganisms as water will leave their cells by osmosis
    • Smoking – smoke contains antibacterial chemicals and the food develops a hardened, dry outer surface.
    • Pickling – an acidic pH kills microorganisms by denaturing enzymes and other proteins.
    • Irradiation – ionising radiation disrupts the DNA structure of microorganisms so they are killed.
    • Cooling/freezing – although microorganisms aren’t killed, the activity of their enzymes is slowed so their metabolism/growth/reproduction is also slow.
  • Using microorganisms to make food (e.g. cheese/yoghurt/bread/alcohol/single cell protein (Quorn – a mycoprotein)
    • Advantages:
      • Can be much faster than animal/plant production
      • Production can be increased/decreased according to demand
      • No animal welfare issues
      • Good source of protein for vegetarians
      • Protein contains no animal fat or cholesterol
      • SCP could be combined with removal of waste products, as they grow on almost any organic substrate e.g. paper or whey
    • Disadvantages:
      • People might not want to eat fungi or food grown on waste
      • Need to isolate protein from the microorganisms in the fermenter
      • Protein needs purification so it’s not contaminated
      • Need to control infection – the microorganisms grow at the same temperatures as harmful pathogens
      • Palatability – the protein has a different taste/texture to meat

How are proteins synthesied from DNA?

The video above is a really good animation to help you visualise what is happening 100 trillion times per second in your body. The process can be split into two main parts; transcription and translation, which are summarised below.

Transcription:

transcription

  1. DNA-helicase unzips the DNA, exposing it.
  2. The exposed base sequence is used as a template for free RNA nucleotides. Activated RNA nucleotides (ones with 2 extra phosphoryl groups)  temporarily hydrogen bond onto the template strand of DNA (leaving the coding strand unchanged). RNA polymerase catalyses this reaction, and the extra phosphoryl groups are released, producing energy for the bonding of adjacent nucleotides.
  3. The mRNA, which is a copy of the coding strand (with T replaced by U), passes out of the nucleus, via a pore in the nuclear envelope, to a ribosome.

Translation:

translation

  1. The mRNA molecule binds with a ribosome. Two codons are attatched to the smaller subunit of the ribosome, and are therefore exposed to the larger subunit. The first codon is always AUG, and so a tRNA molecule with anticodon UAC and amino acid mathionine hydrogen bonds to the codon.
  2. A second tRNA molecule with a different amino acid and complementary anticodon binds to the second codon.
  3. The two adjacent amino acids are joined together by a peptide bond, the reaction being catalysed by an enzyme in the small ribosomal subunit.
  4. The ribosome moves along the mRNA, and a third tRNA molecule brings another amino acid, whoch forms a peptide bond with the dipeptide. The first tRNA is then released to bring another amino acid to the ribosome.
  5. The polypeptide chain continues to grow in this way until a stop codon is reached. The stop codon works because there is no tRNA for the codons UAA, UAC or UGA.
  6. The polypeptide is released and assumes its secondary and tertiary structure.

Collagen Vs. Haemoglobin

Here are the similarities and differences between the structure and function of haemoglobin (as an example of a globular protein) and collagen (as an example of a fibrous protein):

  • Haemoglobin’s quaternary structure is four polypeptide subunits (2 alpha and 2 beta chains) whereas Collagen’s structure is 3 polypeptide chains wound around each other like rope.
  • Haemoglobin has a prosthetic group – each chain contains a haem group (Fe2+) but collagen has no prosthetic group.
  • Haemoglobin is made of a wide range of amino acid constituents in its primary structure whereas approx 35% of collagen’s primary structure is just one type of amino acid – glycine.
  • Much of a Haemoglobin molecule is wound into alpha helix structures but collagen’s molecule mainly consists of left-handed helix structures.
  • Haemoglobin’s function is as a transport molecule and carries oxygen around the body. In contrast collagen’s function is as a structural molecule and to give strength to many cells including artery walls, tendons, bones, and cartilage. This is why it is important that Haemoglobin is soluble (so it can travel around the body in the blood) and Collagen is insoluble (wouldn’t be able to provide support otherwise as would always dissolve).  Haemoglobin’s shape is important so that red blood cells become biconcave disks which can easily travel around the body and bind with oxygen.
  • Haemoglobin is a globular protein meaning its 3D feature is to roll up into balls, yet collagen is a fibrous protein so its 3D feature is to form fibres.
  • Haemoglobin molecules to not bond with each other. However, collagen molecules form covalent bonds between molecules called cross-links which are staggered along the collagen molecules, both increasing strength and forming a fibril. Many fibrils joined together make a collagen fibre.

Haemoglobin:

Collagen: