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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