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DNA Technology- Cloning, Libraries,
and PCR
14 and 16 November, 2005
Text Chapter 20
Cloning Overview
DNA can be cloned
into bacterial
plasmids for
research or
commercial
applications.
The recombinant
plasmids can be
used as a source of
DNA or, if a few
rules are followed,
can be used to
express protein
from any organism.
Restriction enzymes cut DNA at
specific sequences. Many
restriction enzymes leave sticky
ends - ends with single-stranded
regions that are able to form base
pairs with a complementary
sequence.
Role of ampR
and lacZ genes
Cloning DNA into
bacterial plasmids allows
the bacteria to serve as
factories, making large
quantities of the plasmid
of interest.
Start with a number of colonies, each carrying a plasmid with a different
DNA fragment. A radioactive probe can be used to identify colonies
that carry a plasmid that has an insert that is complementary to the probe.
The single-stranded probe base pairs to any plasmid DNA that has
complementary sequence. The fact that it is radioactive makes it easy to
see where it went.
A cDNA contains only
sequence that codes for
protein.
DNA Libraries
A library is a set of
clones that carry
different fragments,
representing the entire
genome of an organism
(genomic library) or
the mRNA expressed
in a certain cell type at
a certain time (cDNA
library).
Libraries can be
constructed in plasmid
or phage vectors.
Electrophoresis
Agarose Gel
Electrophoresis separates
DNA fragments based on
their size. DNA
fragments are often
detected using
fluorescence.
Agarose gel electrophoresis
can be used to investigate an
individual’s genotype directly. If
two alleles have sequence
differences that change a restriction
enzyme recognition site, then the
size differences of the DNA
fragments from a restriction digest
can tell the researcher which alleles
an individual carries.
If this experiment is done on
genomic DNA, then a radioactive
probe complementary to this region
is used to distinguish these
fragments from the rest of the
millions of fragments resulting from
a digest of the genome.
The altered restriction site that produces the different sized
fragments (an RFLP marker) does not have to be in the allele of
interest. It simply has to be closely linked.
The Polymerase chain
reaction can make a large
number of copies of a
specific sequence. The
PCR reaction includes:
•Template DNA
•DNA Primers
•DNA Polymerase
•DNA monomers
The PCR is often used to
answer the same question
that is answered by a
radioactive probe - is a
certain sequence present or
not? If the sequence in
question is present, a PCR
product is made.
Southern Blotting
Determination of DNA
sequence allows the
researcher to determine
genotype at the most
fundamental level - the order
of bases along the DNA
molecule.
This method uses DNA
polymerase to synthesize
new DNA strands in the
presence of dideoxy
nucleotides. Since these lack
a 3’ OH group, whenever one
is incorporated into the
growing strand, that
molecule does not elongate
further.
Genome Sequencing Strategies
Early Conclusions from Genomics
Assembly, annotation and prediction of genome sequence is
computer-intensive. The pattern recognition and minimization
algorithms are ideally suited to vector or SIMD hardware.
Humans have far too few genes - about 30,000. Anternative splicing
is important. The average gene is spliced in two or three different
ways.
Genetic similarity between organisms is striking. Predictions of
relatedness based on morphology are sometimes upheld, challenged
in other cases.
Study of gene expression proceeds on a global level.
Microarray
Hybridization
How was this experiment carried out? What are the conclusions?
Investigating the genotype of individuals can answer questions about
phylogeny (relatedness).
Liquify mite
Purify DNA
PCR
Mt COI gene