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Lecture 14 – Gene Cloning Based on Chapter 08 Genomics: The Mapping and Sequencing of Genomes Copyright © 2010 Pearson Education Inc. 1. Introduction 2. DNA Cloning 3. Restriction Enzymes 4. General Properties of Restriction Enzymes 5. Frequency of Occurrence of Restriction Sites in DNA 6. Restriction Sties (examples) 7. Creation of Recombinant DNA Molecules 8. Cloning Vectors – Plasmid Cloning Vectors 9. Inserting DNA into a Cloning Vector Types of Vectors Thus far we’ve talked about simple cloning vectors – e.g. pUC19, pBluescript Other vectors that are useful for analysis of cloned DNA: 1. Shuttle Vectors – e.g. pRS426 et al. 2. Expression Vectors 3. PCR Cloning Vectors 4. Transcribable Vecotrs 5. Non-plasmid Vectors 6. BACs 7. YACs Shuttle Vectors Yeast Ura3 promoter Yeast 2m ARS E.coli ampr promoter Yeast Ura3 coding sequence (selectable marker) <font face="Verdana" color="#000066" size="3"> pRS426 GPD 5726 bp E.coli F1 ORI (single copy) E.coli ampr Coding sequence Yeast 3’ terminator Multiple Cloning Site Yeast GPD Promoter E.coli ORI (multicopy) NgoMIV NseI Acc651 KpnI XhoI SalI ClaI HindIII XmaI Expression Vectors Practical Issues for Constructing Clones Using an Expression Vector Transcribable/Translatable Vectors An Alternative to Molecular Cloning: Polymerase Chain Reaction (PCR) Polymerase Chain Reaction PCR Cloning Vectors + DNA Ligase Non-Plasmid Vectors 10. Artificial Chromosomes 11. Bacterial Artificial Chromosomes (BACs) 12. Yeast Artificial Chromosomes (YACs) 13. Genomic Libraries Cloning a Specific Gene • Cloning genes from organisms with sequenced genomes usually involves designing PCR primers to amplify the gene from either genomic or cDNA. – The PCR product is then cloned using a PCR Vector. • To clone a gene from an organism that lacks a sequenced genome, several approaches may be used. • The method selected will depend on the molecular tools available. Finding a Specific Clone Using a cDNA Library Screening a Genomic Library Comparing the cDNA Clone and Genomic Clones DNA Primary RNA Transcript mRNA (cDNA) Identifying Genes in Libraries by Complementation of Mutations