Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing.

The research paper presents the construction and analysis of a cDNA library created from RNA extracted from horse skin tissue, which yielded 313 expressed sequence tags (ESTs) that provide insight into the functions of the skin.

Building the cDNA Library

• The researchers began with the extraction of RNA from the skin tissue of an adult horse.
• This RNA was used to create a library of complementary DNA, or cDNA, which is synthesized from an RNA template via an enzyme called reverse transcriptase.
• An oligo (dT) primer, which binds to the tail of the messenger RNA (mRNA), served to initiate the synthesis of the first DNA strand, ensuring the creation of an expression library that will reflect the genes being expressed in the skin tissue.
• The cDNA sequences were directionally inserted, meaning they were incorporated in a specific orientation, to ensure consistency in subsequent analysis.

Characterizing the cDNA Library

• The researchers noted the library contained roughly 580,000 plaque-forming units, which are essentially individual bacteria that have been infected by a single phage containing the cDNA.
• 99.6% of the phages were determined to be recombinant, indicating successful insertion of the cDNA into the phage’s genome.
• The average size of cDNA insert was about 1.3 kilobase pairs (Kbp).

Generation and Analysis of Expressed Sequence Tags (ESTs)

• By sequencing the 5 prime end (the starting point for transcription) of randomly selected skin cDNA clones, the team obtained 313 ESTs.
• The sequencing was performed on an ABI 377 using a technique known as Big-Dye chemistry, which uses dye-labeled terminator nucleotides for detection during sequencing.
• Each EST was checked for similarity against the NCBI non-redundant protein database to identify possible protein-coding sequences, yielding putative identifications for 206 ESTs.
• Scores were also compared to identify redundant ESTs — those representing the same part of the same gene — revealing that 26% of the identified ESTs were redundant.

Categorization by Function

• The ESTs were sorted based on their predicted function, revealing the most frequently identified functional group as translational protein.