Partial sequence of the equine immunoglobulin epsilon heavy chain cDNA.

The research involves isolating a section of the immunoglobulin E (IgE) heavy chain cDNA in horses, using primers designed based on sequences common in humans, sheep, and rats. After successful extraction of the specific DNA fragment, the researchers cloned, sequenced it, and compared its protein sequence with those from the specified species. They discovered that most of the common amino acids are shared between these species and the horse.

Primer Design and Polymerase Chain Reaction (PCR)

• The researchers based their primer design on well-conserved sequences from humans, sheep, and rats. These primers would be able to latch onto the region of horse DNA that they were interested in and amplify it using PCR.

DNA Fragment and Hybridization

• The PCR resulted in a 500 base pair (bp) DNA fragment. This is the section of horse DNA that the researchers were specifically interested in.
• This fragment was made to hybridize, or bond with a human IgE constant region probe. This is essentially a check to see if the DNA extracted is indeed related to the IgE constant region.

Cloning, Sequencing and Comparison

• The researchers then cloned the DNA fragment, which involves making numerous copies of it. This is done to have enough material for further analysis.
• The sequence of the DNA fragment was then determined. This allows the researchers to have a detailed look at the specific order of nucleotides in the DNA segment.
• From this sequence, a ‘derived’ protein sequence was obtained. This is the sequence of amino acids that the DNA is coding for.
• This protein sequence was then compared with the corresponding sequences in humans, sheep, and mice. This provides insights into their similarity and differences, and enhances understanding of how closely related these species are in terms of their IgE proteins.
• They found that most of the common amino acids in these three species are also shared by the horse. This signals a high degree of evolutionary conservation between these organisms for this specific protein.