Molecular Basis for Relationship between Genotype and Phenotype. genotype. DNA. DNA sequence. transcription. RNA. translation. amino acid sequence. protein. function. phenotype. organism. Making Recombinant DNA: Donor DNA. Genomic DNA: DNA obtained from chromosomes of an organism
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Molecular Basis for
Relationship between Genotype and Phenotype
Making Recombinant DNA: Donor 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.
Both restriction fragments are linear and have sticky ends (in this case).
Linear ds DNA is cut with the same restriction enzyme.
By complementary base pairing, the sticky ends can hybridize.
The result is a recombinant DNA molecule.
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.
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
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
* Circular double-stranded DNA
* Replicates independently of chromosomal DNA
* Selectable markers for transformation
* Phage l - clone DNA up to 15 kb
Vectors for Larger DNA Inserts
Hybrid between l phage DNA and plasmid DNA - can carry inserts 35-kb to 45-kb
P1 Artificial Chromosome (derivative of bacteriophage P1) - can carry inserts 80-kb to 100-kb
Bacterial Artificial Chromosome (derivative of F plasmid) - can carry inserts 150-kb to 300 kb
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.