Detection of blood group genes using multiplex SNaPshot method.

BACKGROUND: The determination of blood group antigens in patients and donors is of primary importance in transfusion medicine. Blood group antigens are inherited and are polymorphic in nature. The majority of polymorphic blood group antigens arise from single-nucleotide polymorphisms (SNPs) in the blood group genes. Many DNA-based assays, such as species-specific polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and microchips, have been described to study variant blood group genes. In this study, the SNaPshot (Applied Biosystems) method was adapted to detect SNPs in 10 common blood group systems. STUDY DESIGN AND METHODS: DNA regions of interest were amplified in multiplex PCR and annealed to specific oligonucleotide probe primers of different lengths. AmpliTaq DNA polymerase extended the primers by adding only a single fluorescent ddNTP to its 3' end and was detected by differential mobility in capillary electrophoresis in a genetic analyzer. Results were analyzed using computer software in SNaPshot default analysis method. RESULTS: Seventeen SNP sites in 29 blood samples, previously phenotyped and/or genotyped, were used to test the accuracy and reproducibility of multiplex SNaPshot assays. The results were compared with the previously analyzed types. SNaPshot analyses predicted the 17 SNP sites accurately for all the 29 blood samples. Both homozygous and heterozygous blood groups were detected with equal confidence. CONCLUSION: Blood group detection by SNaPshot method is a practical alternative to antibody-dependent phenotype prediction. Starting with DNA, this method is fast with a turnaround time of 24 hours with mean reagent cost around $2 per SNP detected.

Expression of complementary RNA from chloroplast transgenes affects editing efficiency of transgene and endogenous chloroplast transcripts.

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

RNA editing in ribosome-less plastids of iojap maize.

In maize chloroplasts, 28 C-to-U editing events have been identified in the transcripts of 14 different genes. The iojap mutant of maize, which lacks chloroplast ribosomes, affords the opportunity to examine whether any chloroplast translation products are required for the editing of any of the 28 sites. Furthermore, the mode of action of the IOJAP protein itself is unknown, so we explored the possibility that homozygous ij1/ ij1 plants are defective in RNA editing. Current knowledge of RNA editing in chloroplasts indicates the existence of site-specific factors responsible for recognizing C targets of editing, but the factors have not yet been identified and their encoding genes are unknown. Our results indicate that all 28 editing sites can be recognized and processed in ribosome-less plastids. Transcripts of rpoB are more abundant and more highly edited in iojap mutants. The editing site in rpl2, which creates the mRNA start codon, is the most severely affected in homozygous ij/ ij plants, but nevertheless exhibits at least 10% editing in all mutant lines. Reduced editing of rpl2 may be an indirect effect of reduced splicing, rather than a defect caused by the iojap mutation. We conclude that neither the IOJAP protein nor chloroplast translation products are required for editing any of the 28 C targets of editing in maize chloroplast RNAs.