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1 Molecular Cloning Methods Mohammad Keramatipour MD, PhD Outline DNA recombinant technology DNA cloning co Cell based PCR PCR-based Some application of DNA cloning Genomic libraries cdna libraries Tehran University of Medical Sciences M Keramatipour 1 M Keramatipour 2 Definitions Recombinant DNA: an artificially i constructed t hybrid DNA containing covalently linked sequences with different origins i Recombinant DNA technology: techniques that allow construction of such hybrid DNA Development of Recombinant DNA Limitations in DNA work Genome size, gene copy number and.. Importance of DNA sequencing encing 1964: sequencing of the yeast alanine trna Limitation in sequencing of DNA molecules No available technique to cut DNA at specific points M Keramatipour 3 DNA cutters DNases: very little sequence dependency Restriction enzymes: 1970 M Keramatipour 4 Restriction Endonucleases Definition Charactristics Naming: Genus, species, order of recovery Restriction Enzyme Restriction endonucleases: Role in bacteria Restriction-modification system Restriction site Ends produced: Cohesive ends Blunt ends M Keramatipour 5 Producing blunt ends Polynucleotide kinase Klenow fragment M Keramatipour 6

2 Recombinant DNA Technology Steps Requirements Timing Issues July 1974: Science Legal Guidelines Feb 1975: Asilomar Conference Center: Monterey, Califonia: Asilomar recommendations July 1976: NIH, and RAC (Recombinant DNA Advisory Committee) Jan 1979: Less restrictive NIH regulations GMAG: Genetic Manipulation Advisory Group, UK M Keramatipour 7 M Keramatipour 8 Definition Methods Cell-based PCR-based DNA Cloning Cell-based DNA cloning DNA Cloning Cell-based DNA cloning Components: DNA Restriction enzyme Vector DNA ligase Replicating cell/ host cell M Keramatipour 9 M Keramatipour 10 Vectors Definition: vector is a small segment of DNA that is used as a carrier of another segment of DNA Commonly used vectors Vectors Essential properties of vectors: Autonomous replication Restriction site Selectable marker M Keramatipour 11 M Keramatipour 12

3 Ligation: Methodology Vector-Insert Ligation Problems: Vector re-circularization Vector dephosphorylation Alkaline phosphatase Polymerase chain reaction (PCR) Principles Steps Primer construction DNA denaturation Annealing Chain extension Requirements PCR-based Cloning M Keramatipour 13 M Keramatipour 14 Some Uses of DNA Cloning Human genes and genome Genome project Gene identification/ mapping Analysis of gene function Genetic approaches to human disorders Pathophysiology Genetic testing Treatment Development of expression systems Production of biologically active agents Recombinant insulin (1977 till 1991), growth hormone Hepatitis B virus vaccine Monoclonal antibodies, protein engineering Other areas: industry (detergents), environment,.. M Keramatipour 15 Human Genes, Genome, & Diseases Recombinant DNA technology & human genome, genes, and ddi diseases Developments towards human genome projects DNA libraries Identification of human genes Gene mapping Studying molecular basis of human disorders Transgenic experiments M Keramatipour 16 DNA Libraries - Definitions DNA library: a collection of DNA clones which is meant to collectively l represent a starting population of DNA Genomic DNA library: the starting DNA is the total genomic DNA from a given cell population o cdna library: the starting DNA is cdna Complementary DNA (cdna): DNA synthesized by the enzyme reverse transcriptase using mrna as a template either experimentally or in vivo M Keramatipour 17 Construction of gdna Libraries Starting material: genomic DNA from easily accessible cells (such as white blood cells) Partial restriction digestion with a 4-bp cutter (such as MboI: : GATC/ every 280 bp in human gdna), why partial digestion? Production of desirably large DNA fragments Random fragmentation: better representation of starting ti DNA Containing clones with overlapping inserts M Keramatipour 18

4 Complexity of gdna Library Complexity: is the number of independent DNA clones in a gdna library Genome equivalents (GE): the number of independent clones/(genome size/average insert size) A A library with a genome e equivalent e of 1 is sca called edone- fold library For example: assume a library from human gdna Average insert size: 40 kb 1 GE = 3000 Mb/40 kb = independent clone So a library with clones has 4 GE, so called fourfold library Libraries with GE > 4 are desirable libraries M Keramatipour 19 Making gdna Libraries -1 M Keramatipour 20 Making gdna Libraries -2 Construction of cdna Libraries Starting materials is total RNA from a specific tissue or specific developmental stage of embryogenesis mrna isolation by binding to oligo(dt dt) attached to sollid sepharose or cellulose matrix Convertion to a double-stranded cdna using reverse transcriptase M Keramatipour 21 Ligation of double-stranded oligonucleotide linkers (adaptors) with a suitable restriction site to each ends of cdna to be used for cloning M Keramatipour 22 Making cdna Libraries Making cdna Libraries Making cdna libraries Principle of nick translation M Keramatipour 23 M Keramatipour 24

5 Strategies for Library Screening Directed recombinant screening Hybridization-based based screening Hybridization probes Radioactive labeled probes Can be traced by exposure to radiographic films and so on Biotinylated probes Recognized by fluorescent-dye-coupled avidin A common method in screening libraries i Principles and steps: Colony Hybridizatoin PCR-based screening Once a DNA sequence is known it is possible to design a specific PCR assay M Keramatipour 25 M Keramatipour 26 Thank you all for listening Any comments? M Keramatipour 27