DNA Probes & DNA Hybridisation (AQA A Level Biology)

• DNA hybridisation is a process that is commonly used in medical diagnostic tests and genetic screening
  • In DNA hybridisation two complementary single-stranded DNA molecules combine through base pairing to form a single double-stranded DNA molecule

• When DNA probes are used in conjunction with DNA hybridisation they can indicate whether specific harmful alleles are present in a DNA sample
• A DNA probe is a short length of single-stranded DNA that has a known base sequence complementary to the specific base sequence of a known allele
  • The probe is usually attached to a radioactive or fluorescent label that indicates its position
  • Part of the base sequence of the harmful allele must be known in order to synthesise the DNA probe using a "gene machine"

• Genetic screening can encourage individuals to make lifestyle choices to help prevent disease or provide them with information for viable treatment options

Using DNA probes to locate specific alleles of genes

• A cell sample is taken from a patient
  • This could be a blood sample, a swab of the inside of the cheek, cells from the umbilical cord or amniotic fluid

• The DNA is extracted from the cell sample and purified
• The test DNA obtained from purification is amplified using the polymerase chain reaction (PCR).
  • PCR is necessary as the cell sample will only produce a very small amount of purified DNA
  • PCR produces identical copies of the DNA

• Restriction endonucleases are used to digest the amplified test DNA
  • This is done because whole DNA molecules are too long to be analysed in a single go

• The resulting restriction fragments are separated using gel electrophoresis
  • The negatively charged DNA fragments move through the pores in the gel, towards the positively charged electrode
  • Smaller DNA fragments are able to move at a faster rate through the pores and so they travel a further distance
  • The fragments separate according to size and charge, producing bands in the gel

• The bands of DNA are transferred to a nylon membrane
• The DNA fragments on the nylon membrane are made single-stranded
  • This is done by breaking the hydrogen bonds between complementary base pairs

•  Labelled DNA probes are added to the nylon membrane
  • These DNA probes have a specific base sequence complementary to that of the harmful allele (it must not be complementary to any normal alleles)
  • As the DNA on the nylon membrane is single-stranded the probes can anneal to any complementary DNA fragments present

• The nylon membrane is washed to remove any excess DNA probes and then processed to reveal the position of any bound DNA probes
  • For fluorescent labels, UV light is used to detect their position and for radioactive labels,  autoradiography is used to detect their position

If there is a complementary DNA fragment present on the nylon membrane then the DNA probe will anneal and remain attached to the nylon membrane. The location of the DNA probe is indicated by the label.

• If the label shows up on any of the restriction fragments present on the nylon membrane, then the DNA in that particular position must be from the harmful allele
• If no labels show up then the test DNA does not contain the harmful allele of the gene
• It is important to take into consideration that this kind of process often only tests for one specific harmful allele. An individual may produce a negative test result for that specific harmful allele but they could have another more rare harmful allele, caused by different mutations in their DNA