- Genome annotation by high-throughput 5’ RNA end determination
Genome annotation by high-throughput 5’ RNA end determination
Genome annotation by high-throughput 5â RNA end determination Byung Joon Hwang, Hans-Michael MÃ¼ller, and Paul W. Sternberg Paul W. Sternberg B.A. degree from Hampshire…
Genome annotation by high-throughput 5â RNA end determination
Byung Joon Hwang, Hans-Michael MÃ¼ller, and Paul W. Sternberg
Paul W. Sternberg
B.A. degree from Hampshire College
Ph.D. degree from M. I. T. under Robert Horvitz
Postdoctoral research with Ira Herskowitz at the U. C., San Francisco
Professor of Biology at the California Institute of Technology and Adjunct Professor of Cell and Neurobiology at the University of Southern California School of Medicine, Los Angeles
Byung Joon Hwang â Post Doc
Hans-Michael MÃ¼ller - Wormbase
Trans-spliced Exon Coupled RNA End Determination (TEC-RED)
Identifies 5 â ends of expressed genes
Can distinguish coding regions from regulatory regions
Useful for identifying genes with alternatively spliced 5 â ends.
Developed in nematodes, but can work for any organism which use a spliced leader sequence (Sarcomastigophora, cndarians, nematodes, acoelomate flatworms and ascidians).
70% of mRNAs have one of two splice-leader sequences (SL1, SL2) trans-spliced onto the 5â end
Spliced-leader sequences are transcribed independently as snRNAâs
Trans-splicing with splice-leader sequences produces single-gene mRNAs
Eliminates the large-scale PCR reactions and gel purification of small oligonucleotides steps found in SAGE protocols.
Since the 5â tags are directionally concentrated, the 5â end of the 5â tags are found next to the first anchor RE cut site.
DNA Sequence Analysis
13 525 5â tags (9 401 with SL1 and 4 124 for SL2), obtained from 800 sequencing reactions, were matched to the genome
Represents 2 159 different sequences, 1 639 for SL1 and 520 for SL2
90% of tags corresponded to unique sites
Of the remaining 10%, 90% matched 2 or 3 sites
To remove false positives they analyzed the just 5â of the 5â tag sites for a conserved splice acceptor consensus sequence
93% of the 5â tag sequences remained true positives
75% of tag sequences matched know 5â end in WS100
99 new genes identified
32 previously unknown operons identified
Method for annotation of 5â end of genes.
This protocol works for only organisms with a splice leader sequence
Sequential concentration method applied to SAGE protocols
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