Single-gene analysis is a targeted genetic testing method.
Within the framework of this procedure, if a hereditary disease is suspected, which is triggered by a single change in a gene, the gene suspected as causal is examined. This is also often done routinely as part of screening examinations.
Indications (areas of application)
- Diagnosis of monogenic diseases e.g. cystic fibrosis.
The procedure
Material needed
- Heparin blood (minimum 1-2 ml)
Preparation of the patient
- Not necessary
Disruptive factors
- None known
The laboratory method
Most often, the sequencing method used in single-gene analysis is the Sanger method, also called chain termination synthesis, where it first comes to the isolation of DNA from the blood. Then, all hydrogen bonds that serve to hold the two-stranded DNA together are broken. After heating at 94-96 °C for one minute, two single strands are present. Next, the so-called primer hybridization occurs, whereby a primer is attached. This takes place at about 60 °C. A primer is an oligonucleotide (oligomers composed of a few nucleotides) that serves as a starting point for DNA-replicating enzymes such as DNA polymerase. After primer hybridization, extension takes place, in which the temperature is raised to 72 °C. This allows the primer to attach to the DNA polymerase (enzymes that synthesize DNA). After an average of 30 cycles, the procedure can be stopped.
Further analysis is performed by capillary electrophoresis in automated sequencers. The result is called an electropherogram. This shows different colors indicating the base sequence.
By this method, mutations involving only a single nucleic base within a gene, also known as point mutations, can be detected.