4.3.2007 - 4.3.2007 You would only be using a cDNA library...

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4.3.2007 You would only be using a cDNA library if you know something about the sequence you are trying to find but that information is not good enough to make primers to make PCR o If PCR does not work because you cannot make good primers you would choose one of the two librarys o Advantage of the cDNA library is it is so much smaller, it has such a small fraction of the sequences compared to the genomic libaray o The sequences in the cDNA library are actually different though because there are no introns, have a poly A tail, and no promoter. Because the cDNA that you eventually get through this screening process is different from the gene, that can come into play in terms of the application o What if you want to clone the gene so that you can express it and get that protein product in bacteria? You choose cDNA library because it doesn’t have the introns and you know that bacteria spliceosomes cannot splice those introns out of mRNAs so the presence of the introns would be a problem that would have had to been dealt with ahead of time. It doesn’t have a promoter on it but it’s a eukaryotic promoter anyways so you would have had to get rid of that as well and use a promoter that was already in the plasmid. o So actually if you want to express a human eukaryotic protein of some kind and PCR doesn’t work the cDNA library will work o What if you want to study the regulation of the gene your trying to clone and PCR didn’t work? And regulation is at the transcription phase You need a promoter and sequences upstream of the promoter sometimes so you need these in tact so you need the genomic library. o What if you want to study splicing and alternative splicing? Genomic library, must have introns o Its not all about what is easier but between the two libraries what you want to do with that sequence matters as well (PCR is definitely easier but it also depends on purpose afterwards) Why did we do this? o We wanted to introduce a human gene into bacteria. Why would we do this? We got an unlimited supply of some molecule in cloning and maybe we just wanted the DNA sequence to manipulate it and mutate it to study it (research application). Maybe we watned unlimited supply of what that gene encodes (may be for research purposes or for industrial (commercial) purposes. Some enzymes can have industrial applications. Some things can be used for vaccines where you need to be injected with a bacteria or virus that has been inactivated. What if the bacterial proteins or viral proteins are cloned and then produced in other bacteria and then those proteins isolated and combined into a vaccine…that is called a recombinant vaccine. Then you know you are not actually getting any infectious partical in your body you just get certain protein parts of it – this is a lot safer and should promote the immune response needed. o
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This note was uploaded on 04/07/2008 for the course BIO 325 taught by Professor Saxena during the Spring '08 term at University of Texas at Austin.

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4.3.2007 - 4.3.2007 You would only be using a cDNA library...

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