Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC

Abstract

Under the premise what we have known bacterial artificial chromosome(BAC)clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.

Construction and characterization of a cDNA expression library from the endangered Hu sheep

“…Since cDNAs are produced from fully transcribed mRNAs and contain only the genes expressed of an organism, cDNA libraries are a powerful and useful tool for efficiently identifying gene products (Yang et al, 2012). The construction and analysis of cDNA libraries are considered to be an indispensable tool for functional genomic analysis since these libraries provide considerably detailed information on the genomic mechanisms underlying diverse processes of an organism (Hu et al, 2009;Shao et al, 2009).…”