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Transcript
Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence Making Recombinant DNA: Donor DNA Genomic DNA: DNA obtained from chromosomes of an organism Complementary DNA (cDNA): double-stranded DNA version of mRNA obtained by reverse transcription Chemically Synthesized DNA: DNA sequence obtained by automated chemical reactions Cutting DNA: Restriction Endonucleases Formation of a recombinant DNA molecule Circular ds DNA is cut with one restriction enzyme. Linear ds DNA is cut with the same restriction enzyme. Both restriction fragments are linear and have sticky ends (in this case). By complementary base pairing, the sticky ends can hybridize. The result is a recombinant DNA molecule. Inserting a gene into a recombinant DNA plasmid Vector is a cloning vehicle. Both vector and donor DNA are cut with the same restriction enzyme. Restriction fragments are mixed; sticky ends hybridize. Recombinant vector is the result. DNA ligase seals gaps by forming phophodiester linkages. How amplification works Recombinant vectors are introduced into bacterial host cells. Replication and cell division produce many copies of the recombinant vector. Clones of donor DNA fragments result. Choice of Cloning Vectors: Criteria Small Size: Convenience of manipulation Capability of Prolific Replication: Ease of amplification of donor DNA fragment Convenient Restriction Sites: Single location for insertion of donor DNA Ease of Identification: Quick recovery of recombinant DNA Examples of Cloning Vectors Bacterial Plasmids: * Circular double-stranded DNA * Replicates independently of chromosomal DNA * Selectable markers for transformation Bacteriophages: * Phage l - clone DNA up to 15 kb Vectors for Larger DNA Inserts Fosmids: Hybrid between l phage DNA and plasmid DNA - can carry inserts 35-kb to 45-kb PAC: P1 Artificial Chromosome (derivative of bacteriophage P1) can carry inserts 80-kb to 100-kb BAC: Bacterial Artificial Chromosome (derivative of F plasmid) can carry inserts 150-kb to 300 kb YAC: Yeast Artificial Chromosome - can carry inserts larger than 300-kb Modes of delivering recombinant DNA into bacterial cells (a) Plasmid DNA is introduced into host cell by transformation. (b) Fosmids are introduced in phage heads by transduction. Once inside, they replicate as large plasmids. (c) Phage vectors are introduced by infection.
