26 29 Oct 2010

26 29 Oct 2010 - Foreign genes have variable levels of...

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Foreign genes have variable levels of expression and need to be purified away from host proteins Affinity tags can be fused to the foreign protein by joining the two gene sequences The most common is polyhistidine which has high affinity for metals
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Fusion proteins are easy to retrieve using affinity chromatography The polyhistidine tails bind to metals coordinated on solid supports Maltose binding protein and chitin binding protein are high affinity lectins When fused to a protein of interest, the lectins can bind to column with sugars covalently attached, aiding in the retrieval of the protein of interest
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The use of BACs and YACs allowed for mapping of genomic DNA The DNA sample could be amplified simply by growing the bacteria or yeast and isolating the DNA Searching for genes became easier and neighboring sequences (contigs) could be identified using sequence tagged sites (probe sites)
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In eukaryotes, the genes are spread out over large spans of DNA Each gene has multiple exons with large intervening introns Sequencing contigs then reassembling the gene (identifying exons) is tedious and inaccurate Using a protein isolated from an RNA virus, reverse transcriptase, the mature mRNA can be converted to DNA A genes-only library of copy DNA (cDNA) are available Need a gene – order the cDNA library Need to find a gene – use a probe
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26 29 Oct 2010 - Foreign genes have variable levels of...

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