Labelled DNA probes and DNA hybridisation can be used to find out whether a person carries a particular allele, such as a mutated allele that causes a genetic disorder.1. The base sequence of the allele is found by DNA sequencing.
2. A single strand of DNA complementary to part of the allele is made, and many copies are produced using PCR.
3. A fluorescent or radioactive label is attached, making the DNA probe.
4. DNA is extracted from the person's cells (for example from blood), cut into fragments with restriction endonucleases and heated to separate the strands.
5. The probe is added and the mixture cooled. If the allele is present, the probe hybridises with it.
6. Unbound probe is washed off and the label is detected. Fluorescence or radioactivity shows the allele is present.Often the DNA fragments are first separated by gel electrophoresis and transferred to a nylon membrane before the probe is added, so the band the probe binds to can be seen.
DNA microarrays allow many alleles to be tested at once. A glass slide has thousands of different probes fixed in known positions. In this case it is the person's DNA that is labelled with a fluorescent tag. The DNA is broken into fragments and washed over the slide. Where it hybridises with a probe, that spot fluoresces, and a scanner records which alleles are present.Worked example: a microarray has probes for three alleles of a gene. Spots 1 and 3 fluoresce, spot 2 does not. The person carries alleles 1 and 3, so they are heterozygous.
2. A single strand of DNA complementary to part of the allele is made, and many copies are produced using PCR.
3. A fluorescent or radioactive label is attached, making the DNA probe.
4. DNA is extracted from the person's cells (for example from blood), cut into fragments with restriction endonucleases and heated to separate the strands.
5. The probe is added and the mixture cooled. If the allele is present, the probe hybridises with it.
6. Unbound probe is washed off and the label is detected. Fluorescence or radioactivity shows the allele is present.Often the DNA fragments are first separated by gel electrophoresis and transferred to a nylon membrane before the probe is added, so the band the probe binds to can be seen.
DNA microarrays allow many alleles to be tested at once. A glass slide has thousands of different probes fixed in known positions. In this case it is the person's DNA that is labelled with a fluorescent tag. The DNA is broken into fragments and washed over the slide. Where it hybridises with a probe, that spot fluoresces, and a scanner records which alleles are present.Worked example: a microarray has probes for three alleles of a gene. Spots 1 and 3 fluoresce, spot 2 does not. The person carries alleles 1 and 3, so they are heterozygous.