Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

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.

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.

Inserting a gene into a recombinant DNA plasmid

DNA ligase seals gaps by forming

phophodiester linkages.

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.

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 - clone DNA up to 15 kb

Vectors for Larger DNA Inserts

Fosmids:

Hybrid between 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.