Birds have two sex chromosomes known as Z and W. These are similar to the X and Y sex chromosomes in humans – each inherited from one parent. Male birds have two copies of the Z chromosome (ZZ) and females have one copy of the Z chromosome and one copy of the W chromosome (ZW). This is the opposite of the situation to humans, where most biological females have two copies of the X chromosome (XX) and most biological males have an X and a Y chromosome (XY).
This PCR workflow involves the CHD1 (Chromodomain Helicase DNA Binding Protein 1) genes, which are present on the W and Z sex chromosomes of birds as two variants – CHD1-W (on the female-specific W chromosome) and CHD1-Z (present in males and females on the Z chromosome). These genes are homologous (almost identical in structure and function) but contain introns (DNA that is removed by RNA splicing before it is translated into mature RNA) that usually differ in length between sexes within a species and between species.
This difference in length allows both sexes to be determined using a simple assay based on intron length differences. Once DNA has been extracted, regions within the CHD1-Z and CHD1-W (if present) genes can be amplified using the polymerase chain reaction (PCR), and the amplified DNA visualised on an electrophoresis gel. If only one band is present, this suggests that only one CHD1 coding variant is present (CHD1-Z), which would be expected for a male with a ZZ chromosome pair. If two bands are present, this suggests that both CHD1-Z and CHD1-W genes are present, which would be expected for a female with a ZW chromosome pair.
The advantage of amplifying both variants (if present) together is that the band produced by the CHD1-Z should always be visible – it will only be absent if the DNA extraction or PCR fails. It therefore acts as an individual positive control for each specimen.