Chapter 19
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Part 4: Genomic Analysis
Recombinant DNA Technology
Review
A
enzyme
binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction
fragments.
Most recognition sequences are
, and restriction enzymes can cleave these sequences in an
offset
manner to produce
ends.
DNA fragments can be
using restriction
enzymes
and
such as plasmids and then
replicated
in host cells.
Other
that can carry larger target DNA include
phages,
cosmids,
and
artificial
chromosomes
(BACs).
Often the cloned gene of interest should be
into proteins.
vectors
are engineered to express large quantities of the encoded protein.
Eukaryotic hosts such as
cells
are useful to produce eukaryotic proteins since they can perform
modifications and carry very large DNA fragments.
Bacterial
plasmids
can be used to transfer genes to plant cells in a process caled
.
YACs can be used to transfer genes to animal cells in a process caled
direct
.
The
chain
reaction
(PCR) can be used to amplify small quantities of target DNA
in vitro
.
Genes from whole chromosomes can be cloned to form genomic
. The individual chromosomes can be isolated by
cytometry
and pulsed-field gel
electrophoresis.
Libraries of transcriptionally active genes in particular cells can be constructed from
DNA
(cDNA).
Specific clones can be recovered from a library by
with a
probe
followed by
.
Specific DNA sequences can be identified with a
blot
and autoradiography.
DNA fragments can be
sequenced
by
chain
termination.
Large-scale genome sequencing can be automated by
using
dideoxynucleotides.
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