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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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.

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.