Single Molecule Profiling of Alternative Pre-mRNA Splicing

December 16, 2003

傅瓊玲

Abstract

Alternative splicing is a complex mechanism responsible for gene expression regulation and protein diversity, however, its combinatorial and comprehensive isoforms derived from a single gene are not easy to quantitated and monitoring. Hence, the authors propose a new technology called “digital polony exon profiling” which combines digital nature of PCR and in situ polymerase amplification from a single molecule to study pre-mRNA splicing. 3 examples are displayed to exhibit the power and ability of digital polony exon profiling. MAPT is distinguished six isoforms unambiguously. SMN1 and SMN2 are paralogs with identical protein sequence and different by 5 bases. A technology “SBE” on single-base resolution is applied to SMN’s exon profiling to detect one single base change(C->T) on exon 7 which leads an inefficient inclusion of exon 7 in SMN2, supporting the previous study that different degree of severity caused by SMNs. This technology also applies to CD44’s extracellular domain which involves 10 alternative spliced exons. 69 distinct isoforms are identified even with rare signals and the 15 most abundant isoforms occupy ~95% of CD44 transcripts containing at least one extracelluar exon. This technology is able to monitor the cis relationship of multiple alternative spliced exons within individual transcript and furthermore, be carried out in genotyping, haplotyping and FISSEQ.

from Science. Vol. 301, 836-838. August 2003.

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