Molecular Basis for

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Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
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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
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Cutting DNA Restriction Endonucleases
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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.
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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.
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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.
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Choice of Cloning Vectors Criteria
Small Size Convenience of manipulation Capab
ility 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
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Examples of Cloning Vectors
Bacterial Plasmids Circular double-stranded
DNA Replicates independently of chromosomal
DNA Selectable markers for transformation Bact
eriophages Phage l - clone DNA up to 15 kb
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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
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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.