Download from swjsxy.swu.edu.cn - 西南大学生物技术学院.pptVIP

Section J Analysis and application of cloning DNA Section J -Analysis and application of cloning DNA J1 CHARACTERIZATION OF CLONES J2 NUCLEIC AND SEQUENCING J3 POLYMERASE CHAIN REACTION J4 ORGANIZATION OF CLONED GENES J5 MUTAGENESIS OF CLONED GENE J6 APPLICATIONS OF CLONING J1 CHARACTERIZATION OF CLONES Characterization Determining various properties of recombinant DNA molecule, such as size, restriction map, orientation of any gene present and nucleotide sequence, constitutes the process of clone characterization. It requires a purified preparation of the cloned DNA. Restriction mapping Digesting recombinant DNA molecules with restriction enzymes, alone and in combination, allows the construction of a diagram(restriction map) of the molecules indicating the cleavage position and fragment sizes. Partial digestion The partial digestion of end-labeled DNA fragments with restriction enzymes, and sizing of fragments produced, also enables a restriction map to be constructed. Labelilg nucleic acid DNA and RNA can be end-labeled using polynucleotide kinase or terminal transferase. Uniform labeling requires polymerases to synthesize a complete labeled strand. Southern and Northern blotting The nucleic acid in lanes of a gel is transferred to a memebrane, bound and then hybridized with a labeled nucleic acid probe. Washing removes nonhybridized probe, and the membrane is then treated to reveal the bands produced. Specific RNA species are detected on Northern blots, whereas the DNA bands on Southern blots could be genes in genomic DNA or parts of cloned genes. Fig. 1. Restriction mapping (a) using single and double digests to completion; (b) by partial digestion of an end-labeled molecule. * is the labeled end Fig. 2. 5′-End labeling of a nucleic acid molecule. Fig. 3. Southern blotting. J2 NUCLEIC AND SEQUENCING DNA sequencing The two main methods of DNA sequencing are Maxam and Gilbert chemical method in which end-labeled DNA is subjected to base