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DNA TechnologyCloning, Libraries,
and PCR
17 November, 2003
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.
The restriction-fragment length experiment we looked at before
could use PCR instead of a radioactive probe. If we amplify large
quantities of the region of interest from a small amount of genomic
DNA, and then do the restriction digest, the fragments we are interested
in will be the only ones on the gel.
The other main use of the PCR is in amplifying very small
quantities of DNA. This is useful for forensic investigators, as DNA
from a single cell left at a crime scene can be used as a PCR template
to amplify DNA regions that will indicate who the cell belongs to.
If investigators look at the restriction fragment patterns of a
number of individuals, then they can identify who the cell belongs to.
Since PCR can amplify DNA from a single cell, it is important
to use lab practices that eliminate the possibility of any DNA from the
individuals in question entering the sample from the crime scene.
How was this experiment carried out? What are the conclusions?
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
Investigating the genotype of individuals can answer questions about
phylogeny (relatedness).
Liquify mite
Purify DNA
PCR
ITS regions
(highly variable)