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CSB349 Lecture 10 October 18 th , 2010 Midterm covers everything including this week. Topic today: Molecular genetic and genomic techniques: large scale gene expression analysis Now focusing on RNA transcript Today we’re going to focus on the techniques to globally measure the RNA transcripts. Identification and modification. Regulation comes later. Transcriptome is a collection of all the transcripts produced by the genome. Second Ome after the genome. Gene expression profiling (transcriptomics), take a snapshot of cell and characterise the set of transcripts in the cell at a particular time and place. .which tissue and age, etc. We want to know what are the RNAs present in the particular cell and how many are present. There is a quantitative level of RNA we analyze. The direct and traditional way to is to take all RNAs, make cDNAs, clone and sequence them. .so we make a cDNA library and we understand which RNAs are in the cell, what are the exons. The modern techniques are different. First one is SAGE (serial analysis of gene expr.), sequence only one part of the gene (only good if we know the genome). .ege. a small 12bp fragment of the gene (a good enough identifier). So it’s cheaper and efficient. 12 bp tag is long enough to be unique in the genome. B/c we’re only sequencing 12bps, we can glue them together and sequence them in a row.
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CSB349 Lecture 10 October 18 th , 2010 How SAGE works: Start w/ a tissue, get some cells. Instead of entire cDNAs, you get small pieces (tags), group the tags and sequence all of it, based on the presence of the tag, you can infer the presence of the gene. You get a bunch of tag and you can get relative abundance and know which genes are present and which aren’t. How do you get these tags??: This is done on a bead. Beads w/ oligoDT on them. The OligoDT will hybridize to polyA tail of mRNA transcripts. Convert to ds cDNA. . you just have it attached to beads. Chop some part off w/ a four-cutter (get small four bps size pieces). Then you ligate onto the alul1 cut, a special piece of oligo which you prepare which has Alu1 restriction. You have special restriction site. The restriction enzyme cuts a certain
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CSB349 Lecture 10 October 18 th , 2010 number of base pairs away of consensus site. That means it will cut somewhere inside the cDNA so you
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