HAU16008-04-5 - BIOTECHNOLOGY 160, W2008 Table of...

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BIOTECHNOLOGY 160, W2008 Table of approaches to plant gene cloning (intended as a reference, not intended for memorizing!!) Approach Needed starting information or material Method Comments 1. Map-based cloning beginning with phenotypic character ; also known as “positional cloning” (i) plant lines derived from a genetic cross in which the character of interest is segregating as a single dominant locus (ii) DNA markers that distinguish the parental lines used for the cross (iii) usually, detailed genetic map and/or BAC ( bacterial artificial chromosome ) genomic library for the plant line expressing the phenotypic character Divide the plant lines into two groups: displaying or not displaying the phenotypic character. Attempt to discover specific DNA markers associated with the phenotypic character and as close together as possible on each side of the phenotypic character. Localize gene on a BAC using DNA markers and sequence the DNA region. Common approach but very arduous and time consuming; gene identity must be confirmed, e.g. by complementa- tion . 2. Cloning by complemen- tation beginning with protein function (i) surrogate “organism,” usually E. coli or yeast mutated in gene of interest, plant hairy root or transient expression system, rarely a whole plant, in which the expressed plant protein can be expected to function (ii) selection or screen for the function (iii) in the target plant, a cDNA expression library (each clone has most of the protein coding sequences from polyadenylated RNA plus promoter, etc.) derived from the plant Transform the expression library into the surrogate organism. Apply the selection or screen to identify member(s) of the library in which the plant-derived protein provides the desired function. Recover and determine the cloned sequence from the surrogate organism. Identify the corresponding sequence in DNA from the plant and obtained corresponding genomic clones . Depends on the strong background of molecular genetics available for E. coli and yeast or efficiency of hairy root or transient system. 3. Cloning by protein interaction , beginning with protein function (binding) (i) Gene for a protein (the “ bait ”) with which the target plant protein is postulated or known to interact (ii) two-hybrid, ” “ phage display ” or other selection system to detect binding of other (“ prey ”) proteins to the bait protein
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This note was uploaded on 12/03/2010 for the course ECONOMY Eco 100 taught by Professor Taidat during the Spring '10 term at Hanoi University of Technology.

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HAU16008-04-5 - BIOTECHNOLOGY 160, W2008 Table of...

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