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Lecture CSB349 10 Midterm covers everything including this week. October 18th, 2010 Topic today: Molecular genetic and genomic techniques: large scale gene expression analysis Now focusing on RNA transcript Today were 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 its cheaper and efficient. 12 bp tag is long enough to be unique in the genome. B/c were only sequencing 12bps, we can glue them together and sequence them in a row. CSB349 Lecture 10 October 18th, 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 arent. 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 CSB349 Lecture 10 October 18th, 2010 number of base pairs away of consensus site. That means it will cut somewhere inside the cDNA so you get a fragment that has you linker. Now instead of having whole cDNA, you just have a tag. Then you get ditags (each half corresponds to a sequence of whichever the ns are), What is Alu1???? Used for measuring copy number in the genome. You can quantitively monitor the expression number of thousands of gene simultaneously. Two major kinds of microarrays: one is traditional. The more modern thing is a DNA chip...they are more condensed and can fit even more probes onto the DNA strip. They are a substrate where you immobilize on here DNA probes. CSB349 Lecture 10 October 18th, 2010 How does it work? You have two samples you want to measure their transcriptome. You compare them to eachother. Traditional always involves two. Fibroblasts with serum or without. You do a trick, you make cDNAs where you incorporate fluorescent labels. The trick is you do this with two different colours. One for one sample, one to the other. Green and Red. Labelled cDNAs, mix two samples together and spill them onto a microarray and let it sit to get it to hybridize and get probes on the array to hybridize with one of your labelled cDNAs if its present. Each probe corresponds to a gene. Each spot has millions of copiues of probes. Each spot has many probes so there is plenty of opportunity for them to hybridize on. Then scan it using a fluorescent scanner. Then you measure the levels of the fluorescence intensity. Most of the spots you see will be yellow. Most will be same amounts of spots of cDNA. Blue spots are not possible so ignore them. Scanner quantitates the fluorescence. CSB349 Lecture 10 October 18th, 2010 Level of fluorescence instensity is proportional to the levels of transcripts. You have a limited number of probes on your microarray..you can actually saturate it with too many transcripts...no more room or you reach a limit where your scanner cannot detect anymore bright fluorescence. You assume the hybridization signal you see corresponds to the amount of labelled cDNAs hybridized there but that may not be true. Thousands of probes used at same time so its difficult to check every spot. You need to be very in careful designing the area otherwise you....it may be difficult to design a probe that actually distinguishes b/w CSB349 Lecture 10 October 18th, 2010 two gene families (or two things w/in gene family???) positive and negative controls needed. How can you design a control thats going to work with all the thousands of transcripts. Now that youve measured all these levels of DNA, how do you analyze? Takes too long to do it one at a time...there are statistical methods. There is cluster analysis...a way to find patterns to find data. Find groups of genes that show similar expression patterns. Typically when you do a microarray, you find genes showing one pattern ...you can compare one cell type to another. Look at entire gene expression pattern of the whole cell. An example of cluster analysis: This is a heat map showing gene expression data for microarrays. Each column is a different time point in a series of experiments a different microarray. The colours indicate the relative changes in gene expression levels. Black = no change. Green = reduction is expression level red=increase in expression levels. ALL OF THESE are CSB349 Lecture 10 October 18th, 2010 only RELAVTIVE changes. Each ROW in this picture represents a single gene, each column is a separate experiment. Cluster analysis organizes the genes such that those that show the same pattern will be in the same area. Form groups that are similar. Usually same family of genes. E.g. Some of the genes here, pull out a group of genes Another way Transcriptomics is used is to identify new genes, their position, etc. DNA chip is used, it has probes across the whole genome and immobilize it on a slide. You can extract RNA from your cell and spill it onto this microarray and look at probes for not only known genes but for novel genes as well. You may see a peak of expression in a new part of the genome where you didnt know a gene was there before. A way of finding thousands of new transcripts. New regions of the genome correspond to places of active transcription. Annotation (give a name to particular place in genome). CSB349 Lecture 10 October 18th, 2010 We can use oligonucleotide arrays to study protein DNA interactions ChIP on ChIP experiement. Not just some promoter to some particular gene. You can use it genome wide. To find which location a specific DNA binding protein is found. Crosslink protein to your DNA, use an antibody specific to that protein to immunoprecipitate DNA and DNA complex, Release the DNA, you only have the proteins you have antibodies to. You make labelled ones and hybridize them to a chip and put it in a scanner and you get relative abundances of chip signals at all these different areas. CSB349 Lecture 10 October 18th, 2010 There are very few green or red spots here. Most are yellow. Yellow = no enrichment, Red= enriched signal, . Two coloured array. Typically youll use as a control the entire genomic DNA w/o IP or you could use a mock IP but you dont put antibody in there , or you can use an antibody to something else (from another species..see if antibody is specific to the protein). The graph is the signal you see comparing your actual IP sample to your control. Each spot represents a single probe in your DNA. How far away from diagonal concludes its an enriched point. Clear red spot on top. The probe is greatly enriched, bottom one is not. CSB349 Lecture 10 October 18th, 2010 Used to identify components of polychrome complex. In order to do it, you need a LOT of cells. You can do it w/ a whole embryo, but not with one cell. Can see how many points (lots), that had enrichment (off of diagonal) Put in DNA that hadnt been enriched, peaks correspond to enrichment. Theres a large region showing signal. RH one is where they took a different antibody (negative control), used against their input. CSB349 Lecture 10 October 18th, 2010 Exact same process used in Chip Chip can be done by sequencing instead of by DNA microarray or gene chip. how new sequencing works (not in test). You do a synthesis of many many fragments on a substrate (glass), you have labelled dyes for each of the four bases .As you synthesize the sequences in vitro on the substrate, you use the modified nucleotides so they release a flash of light as they incorporate onto strand, you take many many pictures as you add them. CSB349 Lecture 10 October 18th, 2010 CSB349 Lecture 10 October 18th, 2010 ... View Full Document

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