15 - Genomic DNA libraries Start with genomic DNA from any...

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Unformatted text preview: Genomic DNA libraries Start with genomic DNA from any cell DNA has to be broken into smaller pieces and ligated into vector Vector just has to be replicated so no translational or transcriptional signals necessary in vector, just replication signal Usually, bacteria used as host cell various vectors can be used (plasmid, phage, cosmid, BAC) genomic DNA libraries genomic library - collection of clones representing the entire genome of an organism vector genomic DNA cut with RE recombinant DNA Involves 3 components: 1) source organism (organism DNA is extracted from) 2) vector (a plasmid would be an example) 3) host organism (organism into which recombinant DNA will be inserted and maintained) Plasmids can be engineered to serve as vectors for recombinant DNA techniques Engineered plasmids: Ori Selectable markers Polylinker region Vector just has to be replicated so no translational or transcriptional signals necessary in vector, just replication signal genomic DNA libraries Why create genomic libraries? genome sequencing projects - sequence all of the DNA fragments and reassemble the sequence data with computer algorithms screen clones for specific DNA of interest - this would be what you would need to do if no sequence data was available for a particular organism. In this case you could not simply PCR the DNA of interest. investigations of regulatory DNA sequences !"#$ &()*+",- ./0 1# 2"23-"4125 6 7)-8 -&99 -"2$)12# $8& #):& -":;9&:&2$ "< ./0= >$ 12 41?&*&2$ -&99 $+;&# 41?&*&2$ #&$# "< 5&2&# )*& #@1$-8&4 "2 @819& "$8&*# )*& #19&2$ Relatively few genes are expressed in any one type of cell If mRNA is extracted and used as starting material, then fewer clones to screen Clones would represent only genes being expressed in that cell type Figure 8.6 cDNA library Start with all mRNAs from a particular cell type or tissue No need to digest have individual mRNA molecules Must make double-stranded cDNA (RNA reverse transcribed back to DNA) and ligate into a vector If you want expression as well (cDNA expression library), vector must have transcriptional and translational signal appropriate to host cell cDNA synthesis (or RNaseH) cDNA synthesis (cont.) cDNA library construction Once you have a cDNA sequence for...
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This note was uploaded on 03/31/2010 for the course BIMM bimm 100 taught by Professor Dr.sato during the Winter '08 term at UCSD.

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15 - Genomic DNA libraries Start with genomic DNA from any...

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