ALBERT

Description: Background High-density oligonucleotide arrays are widely used for analysis of genome-wide expression and genetic variation. Affymetrix GeneChips – common high-density oligonucleotide arrays – contain perfect match (PM) and mismatch (MM) probes generated by changing a single nucleotide of the PMs, to estimate cross-hybridization. However, a fraction of MM probes exhibit larger signal intensities than PMs, when the difference in the amount of target specific hybridization between PM and MM probes is smaller than the variance in the amount of cross-hybridization. Thus, pairs of PM and MM probes with greater specificity for single nucleotide mismatches are desirable for accurate analysis. Results To investigate the specificity for single nucleotide mismatches, we designed a custom array with probes of different length (14- to 25-mer) tethered to the surface of the array and all possible single nucleotide mismatches, and hybridized artificially synthesized 25-mer oligodeoxyribonucleotides as targets in bulk solution to avoid the effects of cross-hybridization. The results indicated the finite availability of target molecules as the probe length increases. Due to this effect, the sequence specificity of the longer probes decreases, and this was also confirmed even under the usual background conditions for transcriptome analysis. Conclusion Our study suggests that the optimal probe length for specificity is 19–21-mer. This conclusion will assist in improvement of microarray design for both transcriptome analysis and mutation screening.

Description: Background The quail and chicken major histocompatibility complex (Mhc) genomic regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated class I, class IIB, natural killer (NK)-receptor-like, lectin-like and BG genes. Therefore, the elucidation of genetic factors that contribute to the greater Mhc diversity in the quail would help to establish it as a model experimental animal in the investigation of avian Mhc associated diseases. Aims and approaches The main aim here was to characterize the genetic and genomic features of the transcribed major quail MhcIIB (CojaIIB) region that is located between the Tapasin and BRD2 genes, and to compare our findings to the available information for the chicken MhcIIB (BLB). We used four approaches in the study of the quail MhcIIB region, (1) haplotype analyses with polymorphic loci, (2) cloning and sequencing of the RT-PCR CojaIIB products from individuals with different haplotypes, (3) genomic sequencing of the CojaIIB region from the individuals with the different haplotypes, and (4) phylogenetic and duplication analysis to explain the variability of the region between the quail and the chicken. Results Our results show that the Tapasin-BRD2 segment of the quail Mhc is highly variable in length and in gene transcription intensity and content. Haplotypic sequences were found to vary in length between 4 to 11 kb. Tapasin-BRD2 segments contain one or two major transcribed CojaIIBs that were probably generated by segmental duplications involving c-type lectin-like genes and NK receptor-like genes, gene fusions between two CojaIIBs and transpositions between the major and minor CojaIIB segments. The relative evolutionary speed for generating the MhcIIBs genomic structures from the ancestral BLB2 was estimated to be two times faster in the quail than in the chicken after their separation from a common ancestor. Four types of genomic rearrangement elements (GRE), composed of simple tandem repeats (STR), were identified in the MhcIIB genomic segment located between the Tapasin-BRD2 genes. The GREs have many more STR numbers in the quail than in the chicken that displays strong linkage disequilibrium. Conclusion This study suggests that the Mhc classIIB region has a flexible genomic structure generated by rearrangement elements and rapid SNP accumulation probably as a consequence of the quail adapting to environmental conditions and pathogens during its migratory history after its divergence from the chicken.