Molecular Cloning and Analysis

Gene cloning
        1. DNA isolation
        2. formation of library
                a) restriction enzymes, ligases
                b) vectors: plasmids  ( 0.1-10 kB)
                                l phages  (10-20 kB)
                                cosmids -COS sites allow packaging into l heads (45kB)
                                YACS- yeast artificial chromosomes  (50-700 kB)
                                BACS- bacterial artificial chromosomes  (100-300 kB)
                                P1 vector-P1 phage (70-95 kB)
                c) types of libraries
                        -genomic library- fragments of genomic DNA
                        -cDNA library- DNA copies of mRNA expressed 
                                -varies with source of mRNA
                                -only expressed sequences, no promoters
                d) screening
                        -hybridization with other cloned gene or from aa sequence
                        -functional complementation
                        -antibody screening
                        -subtractive cloning
                        -(map-based cloning)

                

        3. tricks- 
                Polymerase chain reaction
                        -amplify fragment of DNA based upon known flanking 
                                sequences (e.g. conserved motifs in proteins)

Gene characterization
        Blotting- separate nucleic acids on gel, transfer to membrane, blot with                        radioactively labeled probe DNA
                -Southern blot- shows environment of gene in genome and presence of 
                        related genes
                -Northern blotting- shows size and expression level of mRNA

        Sequencing
        What a cloned gene allows you to do:
                        -sequence allows guess at function
                        -transfer to another organism or express at wrong time to 
                                determine function
                        -follow regulation- what turns it on or off
                        -can alter gene- what parts do what
                        -see how gene interacts with other proteins to determine trait
                        -understand evolution of a trait or relatedness of organisms
				e.g. rbcS -based tree Clegg et al. PNAS 94: 7791

Gene sequence analysis exercise

Links:
Animation of molecular techniques-PCR, Southern blotting, sequencing
Animated PCR and Blotting demo
Cloning vectors, screening, characterization-series of articles from PLSC431 NDSU
Molecular Biology Techniques - MIT Hypertex
Cloning and cloning vectors-PLSC731 NDSU
Basics of Molecular Genetics
Recombinant Techniques- UC Davis BS101 Course lecture
Technical Protocols for DNA Manipulation and Cloning
Protocols for plant molecular biology
P1 vector cloning
gene database searching
Restriction enzyme database
List of sequence databases- abstracts and links to databases
The Tree of Life- Phylogeny, much based upon DNA sequencing
 

Last revised: Feb. 16, 2000- Straney