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!

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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.

The Lac Operon

Explain genetic control of protein production in a prokaryote using the lac operon

Ecoli

E-coli grown without lactose are placed in a medium with lactose. At first they cannot metabolise the lactose as they only have tiny amounts of the enzymes needed to metabolise it. After a few minutes the rate of synthesis of these enzymes increases. Lactose triggers the production of these enzymes and so is the inducer.

the lac operonStructural genes code for the enzyme, the operator region can switch them on and off, and the promoter region is where RNA polymerase binds to begin transcription. The regulatory gene is not part of the operon and may be some distance away.

 lactose absent

When lactose is absent:

  1. The regulator gene is expressed and the repressor protein is synthesised. It has binding sites for lactose and the operator region.
  2. he repressor protein binds to the operator region, covering part of the promoter region where RNA polymerase attaches.
  3. RNA polymerase cannot bind to the promoter region so the structural genes are not transcribed to mRNA, so the genes cannot be translated into the two enzymes.

lactose present

When lactose ispresent:

  1. Lactose (inducer) molecules bind to the other site on the repressor protein, causing the repressor protein to change shape and dissociate from the operator region.
  2. The promoter region is now unblocked so RNA polymerase can bind to it and initiate transcription.
  3. The operator-repressor-inducer system is a molecular switch.
  4. The E. coli can now make lactose permease to take up lactose and β-galactosidase to convert it into glucose for respiration.

Here is an animation which summarises the above:

10 things you’ve always wondered about humans, explained.

 

I_Wonder

1. Why do women test perfume on their wrists?

In order to smell perfume it has to be vapourised using heat, and as there are lots of blood vessels just under the skin surface of the wrist it is a warm part of the body where the perfume will vapourise quickly.

2. Cocaine can enter the bloodstream by being sniffed, so why can’t diabetics inhale Insulin instesd of having to inject it?

To pass into the blood vessels in the nose drugs must pass through the thin epithelium lining the nasal passages, but drugs which are not fat soluble cannot pass through the phospholipid bilayer of cells, and as Insulin is not fat soluble (whereas cocaine is) it wouldn’t be able to pass through the nasal cells to enter the bloodstream.

3. Why have we not selected against and eradicated genes from the human genomw which cause ageing?

In order for genes to be passed on, the orgnism must reach reproductive age, so any characteristics, and therefore genes, which help the organism to survive so it can breed will mean the organism is more lkely to survive and pass those genes on. However, genes which cause ageing have no affect upon the survival of the species as these characteristics are only expressed after reproduction.

4. Why does one year seem longer to a child?

Time is relative. A child of 10 will think a year is very long as it is 10% of all the time they’ve lived so far, but to a 50-year-old one year is only 2% of  all the time they’ve lived, so as one year is a much larger proportion of a child’s life it appears much longer to them, and this is also why time appears to speed up as we get older.

5. If humans are so sophisticated why to we only have 46 chromosones when some plants such as ferns have around 1320?

Numbers of chromosones have nothing to do with sophistication, rather they are indicator of the age of the species. As ferns have been evolving for millions of years they have experienced more mutations in their chromosones which has led to an increase overall in the number of chromosones.

6. Why is milk white when tears and sweat are colourless?

It is simply down to the fact that milk contains fat forming a white-coloured emulsion whereas sweat and tears only contain mainly water and salts.

7. Why do our shins, which often suffer when we walk into something, have so little protection?

Our ancestors walked on all fours and so their shins would be on their hind legs and were rarely banged into so there was no need to protect them.

8. Why do females of other species, such as dogs, have much less painful pregnancies?

Animals who walk on four legs havethe weight of their offspring shared equally between four legs instead of two, and their backbone provides a horizontal support. As we walk on two legs the weight of the baby pulls forward, which is why many pregnant women experience severe backache.

9. Why do we have two sets of teeth?  

If we only had one set, as the jaw continued to grow during childhood, gaps would appear between our teeth as teeth cannot grow sideways, so we need a second set for when our jaw has stopped growing.

10. Why are more people dying each year from cancer nowadays, depsite huge advances in medical treatment and improving survival rates?

Vaccination has meant that very few people each year die from diseases such TB, polio, pneumonia etc and so as this has greatly increased our life expectancy, and our population is continually increasing,  more people are reaching the older age groups where cell mutations, i.e. cancer, occurs more frequently.

Homeostasis: Maintaining Body Temperature

Key Definitions:

  • Ectotherm: regulates body temp. via external sources e.g. the sun
  • Endotherm: can generate heat internally to regulate body temp.  e.g. metabolism in the liver.
  • Negative feedback: brings a reversal of any change of conditions back to the optimal conditions.

Ectotherms

iguana

Advantages:

• Need less food as less is used in respiration, so can go for long periods of time without food and more energy obtained from food can be used for growth.

Disadvantages:

• At greater risk of predation as less active in cool environments and may need to warm up in the mornings before they can become active.
• May be incapable of activity in winter months so need to build up large energy store so can survive without eating.
 
Ectotherms change their behaviour or physiology to react to environmental temperature changes:
• Expose body to sun to enable more heat to be absorbed
• Orientate body towards sun so larger S.A for heat absorption (or away from sun so less heat absorbed)
• Hide in burrow reducing heat absorption
• Alter body shape to expose smaller/greater S.A to sun
• Increase rate of breathing so more water evaporates
 
Endotherm:
 
welcome and be happy
 
Advantages:
 
• Fairly constant body temp. despite any external temp. changes
•  Can be active when it’s cold i.e. night/morning/winter and so can live in colder parts of the planet

Disadvantages:

• Large proportion of food intake used to maintain body temp, so more food is needed, and less food is used for growth.
 

Physiological adaptations:

• Water can evaporate from lungs/nose/mouth or from the skin when the sweat glands produce sweat
• Hairs on skin can be raised/lowered to trap greater/smaller insulating air layer
• Vasodilation/vasoconstriction in arterioles leading to skin to increase/decrease radiation of heat at skin surface
• Liver cells can alter their rate of metabolism
• Skeletal muscles can contract spontaneously ti generate heat via respiration in muscle cells (shivering)
 

Behavioural adaptations:

• Move into shade/hide in burrow
• Orientate body towards/away from the sun

 Remain inactive and spread out limbs to increase S.A, or roll into a ball to decrease S.A

 

Body temperature in endotherms is controlled by negative feedback:

h2

The body also contains peripheral temperature receptors located at the extremities so there is a quicker reaction to external temp. changes as core body temperature may take some time to decrease enough for the hyperthalomous to detect a significant change.

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.