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