Chapter_6_-_Genomic_and_cDNA_Libraries_v

Chapter_6_-_Genomic_and_cDNA_Libraries_v -...

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6.1 Genomic Libraries In a genomic library, the whole genome is broken into small fragments cloned into vectors There are problems with fragmenting the DNA with a particular enzyme (eg. EcoRI) produces non-overlapping fragments, making it difficult to put together the full gene a wide array of fragment sizes Connection of one clone with another requires a library of overlapping fragments Figure 6.1 Genomic libraries: overlapping and non-overlapping fragments
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6.1 Genomic Libraries 6.1.1 Partial digests partial digestion is one way to construction a library of overlapping fragments Partial digests are created with: Small amounts of enzyme or Short digestion times Digested fragments can be size selected to eliminate cloning of very, small fragments Problems of over or under- representing DNAs with enzyme digestion: Random cuts using: Mechanical shearing (passing DNA through syringe) or ultrasonication Mechanical breaking of DNA produces blunt or ragged ends that have to be filled or trimmed before adaptors/linkers added to the ends Figure 6.2 Using a partial digest to produce a collection of overlapping fragments
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6.1 Genomic Libraries 6.1.2 Choice of vectors Choice of vector influences the size of the insert that vectors can accommodate Size of the insert also affects the size of the library that is necessary to obtain a complete representation of the genome Have to create more than the size of the genome in inserts to have complete representation of the genome Have to search through many genome size worth of clones because the library inserts are random, overlapping and redundant. As increase the size of the library, it is less and less likely that each additional clone adds new information, but also there is no guarantee that all the genetic information of the genome is present in a library Number of independent clones (which
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_6_-_Genomic_and_cDNA_Libraries_v -...

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