MT4 - Metagenomics Cataloging whats there Comparing one...

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Metagenomics Cataloging whats there Comparing one genome to the other Is now more concerned with biological pathways than organisms mRNA Difficult to sequence proteins but not RNA 3 reasons to sequence RNA o To know WHAT the genes are o To know WHERE they are o To know EXPRESSION PATTERNS OF GENES Reverse transcriptase Used to make RNA into DNA since RNA is not the most stable o RNA –DNA hybrid o DNA-DNA >>> cDNA (copyDNA) Requires a template (RNA) and primer (DNA or oligo) cDNA libraries Collection of cDNA’s cloned into plasmids First you need RNA > poly T tail for poly A tail Since priming from the poly A tail usually falls off, people often use random oligo primers for the RT reaction EST’s Expressed sequence tags From the ends of cDNA’s Plasmid vector has known sequence, one can prime from the vector and sequence into the known vector and sequence into the unknown cDNA Can prime from 3’ end or 5’ end 5’ end is better because it is where RT fell off and is a random event 3’ end can tell if the two EST’s come from the same gene If one has a lot of 5’ end EST’s one can even assemble complete transcripts Full length cDNA Sequencing the whole cDNA is like a small shotgun genome assembly where one builds
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