genomic library - use of restriction enzymes (Oct/11/2008 )

1. a genomic library can be made by completely digesting a plasmid vector with BamHI and ligating with chromosomal dna that has been partially digested with Sau3A. why are 2 different enzymes used?

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BamHI is commonly used as a restriction site in cloning vectors. The difference between BamHI and Sau3A is their length in their base-pair recognition site and, consequently, in their frequency of cutting the DNA. BamHI has a 6bp-recognition sequence whereas Sau3A recognizes a 4bp-sequences. So Sau3A will produce smaller pieces of your genomic DNA than BamHI would (because it would cut more often). I guess this all would depend how big you would want your genomic DNA pieces to be in the first place. Both enzymes produce identical cohesive ends and therefore DNA pieces (vector and genomic DNA) can be ligated.

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thank you for responding. i do understand all you outlined but why use a smaller cutter (4-bp), then do partial digestion? (facilitates longer sequences).

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I believe one would use a 4bp-cutter because there are more cutting sites all together on the DNA available (it occurs more frequently). But one would only do it partially because otherwise the fragments would be too short (only about 300bp).

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why not use BamHI to cut Dna instead?

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Could it be to have more genes in recombinant prior to amplification ?

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Here's the main reason.

1. BamHI and Sau3A got the same recognition site as noted earlier
2. An average gene size is around 3kb (For a genomic library it might be different desired size!!)
3. Generally we would aim for a cut size aroun 6kb if our main interset is getting a gene.. (For a genomic library it might be different!!)
4. You can't generate a size small enough with BamHI, you could probably get abit but not as much as you would expect as BamHI is a 6bp cutter, less probability of cutting
5. Too big of a cut size increases the size of insert+vector thus making the transformation hard and less efficient ( NOT a good thing if u want good genomic representation@@)
6. In addition , big insert+vector contain so much genes that it will make subsequent sequencing a pain because you would have to generate more primer of your own..to continue sequencing!

For interest sake ,

BamHI reg site ==> 5'GGATCC 3'

Sau3A reg site ==> 5' NGATCN 3' ,

hope that helps?!

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thanks! you made it crystal clear!

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What is the main reason for using a cDNA library rather than a genomic library to isolate a human gene from which you wish to make large quantities of the human protein in bacteria?

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Unlike bacteria genomic DNA, our genomic DNA is full of repetitive DNA and non coding DNA and plus some of our gene require splicing prior to being fully functional

cDNA is generated from spliced RNA which code for functional gene so therefore, in human or any eucaryote it's better to do a cDNA library.

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