Lecture 22

Lecture 22 - Lecture 22 Molecular Techniques DNA library:...

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Lecture 22 Molecular Techniques DNA library : Collection of DNA molecules each cloned into a vector molecule Genomic library : Set of clones that collectively represent all the DNA sequences in the genome Genomic DNA is the source of starting DNA Advantages : Ideal for representing genetic content of relatively simple organisms such as bacteria or yeast Disadvantages : Difficulties in higher eukaryotes Extensive introns sequences that are too large to insert into a plasmid vector Sequences can be broken and carried into more than one clone Difficult to identify important parts of a gene Protein coding sequences : 1.5% of our genome Cellular mRNA must useful material for generating a DNA library Lacks non coding regions present in genomic DNA cDNAs : DNA copies of mRNAs Synthesized and cloned into plasmid vectors cDNA library : Collection of resulting cDNA clones Preparation of cDNA library through reverse transcriptase I Page | 1
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Isolate the total mRNA from the cell type Poly A is characteristic of mRNA’s Reverse transcriptase: Used to synthesize a strand of DNA complimentary to each mRNA molecule starting from a Poly T primer Resulting molecule is converted to double stranded DNA molecules corresponding to original mRNA Cloning: Double stranded DNA containing recognition for particular restriction enzymes are ligated at both ends Treated with restriction enzyme to generate sticky ends Plasmid DNA is also treated with same restriction enzymes Vector + collection cDNA joined covalently by DNA ligase Transfered to E.coli to generate individual clones Method Page | 2
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1.Isolate mRNA with a Poly (A) tail Tag that makes mRNA different is the Poly (A) tail Easy to fish out from rRNA and tRNA 1.Use poly T as a primer Hybridizes with Poly (A) tail Specifically hybridizes with A 1.Poly T primer used for reverse transcription starts from the tail of mRNA Produces hybrid of DNA+RNA 1.Digestion of RNA with RNases H Not yet digested piece of RNA is used as a
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Lecture 22 - Lecture 22 Molecular Techniques DNA library:...

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