LS18DNALibrary

LS18DNALibrary - DNA library and clone identification What...

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DNA library and clone identification What is a DNA library? A collection of all DNA of a system, such as the genomic DNA of Arabidopsis, or cDNA of a human organ Why need a DNA library? So we can easily isolate a DNA of interest, such as a gene. A library can be permanently stored and used.
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The best way is to clone the DNA fragment into a vector and let a cell replicate the DNA fragment for us. An alternative way to obtain more copies of the same DNA fragment is to use PCR amplification. The PCR products can also be used as an insert to be cloned into a vector for further amplication of the DNA of interest in cells. (1) The first scenario - we have the known DNA fragment, but want to make more of it
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(2) The second scenario - we don't have the gene, and want to isolate it (Cloning of a specific gene) In order to isolate a gene, one needs two things: a “ DNA library (genomic library or cDNA library) that is a collection of randomly cloned DNA of an organism, and a " probe " that is a DNA of interest and it must be labeled one way or another. 1. A “ genomic library ” is a collection of clones (often phage) each contain a piece of genomic DNA of the target organism. Genomic library can be used to isolate any regions of the genome 2. A " cDNA library " is a collection of clones (also phage) of different DNA that are copies of the mRNA. So the cDNA library contains only the transcribed regions of the genome.
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Genomic library and cDNA library
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Comparison of genomic library and cDNA library Genomic library: 1. copies of genomic DNA, 2. contain introns, exons, and un-transcribed regions of the genome including all the ”junk” DNA 3. each gene is presented with same copy number in the library. 4. Genomic libraries made from different tissues of the same organism are almost the same. cDNA library 1. copies of mRNA 2. contain only transcribed region of the genome, no intron or promoter 3. The copy number of each gene in the cDNA library may be different. Highly abundant mRNA species will have more copies in the library 4. cDNA libraries made from different tissues of the same organism can be very different because mRNA expression profiles vary among tissues. 4. A cDNA can be transcribed with a heterologous promoter and translated to protein directly in cells.
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Vectors for cDNA and genomic libraries Depending upon the sizes of inserts and the purposes of libraries, different vectors can be used to construct cDNA and genomic libraries. In general, λ phage and plasmid are usually used as vectors for cDNA libraries, whereas λ phage, BAC and YAC are usually used as vectors for genomic libraries <
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For a genome library to carry all of the possible fragments of a large genome, the transformation efficiency must be high. Human genome = 3x10
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This note was uploaded on 03/14/2012 for the course LS 3 taught by Professor Lin during the Fall '06 term at UCLA.

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LS18DNALibrary - DNA library and clone identification What...

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