8-4-7-dna-probes-and-dna-hybridisation

DNA probes & DNA hybridisation

• 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

• 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

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

• When DNA probes are used in conjunction with DNA hybridisation, they can indicate whether specific harmful alleles are present in a DNA sample

  • Part of the base sequence of the harmful allele must be known in order to synthesise the DNA probe using a “gene machine”

Using DNA probes to locate specific alleles of genes

1. 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

2. The DNA is extracted from the cell sample and purified

3. The test DNA obtained from purification is amplified using the polymerase chain reaction (PCR).

4. 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

5. The resulting restriction fragments are separated using gel electrophoresis

6. The bands of DNA are transferred to a nylon membrane

   • The DNA fragments on the nylon membrane are made single-stranded by breaking the hydrogen bonds between complementary base pairs

7.  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

8. 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

   • For radioactive labels, autoradiography is used to detect their position

Interpreting results

• 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 rarer harmful allele, caused by different mutations in their DNA