DNA Technology- Cloning, Libraries, and PCR 14 and 16 November, 2005 Text Chapter 20

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DNA Technology- Cloning, Libraries, and PCR14
and 16 November, 2005Text Chapter 20
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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.
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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.
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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.
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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.
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A cDNA contains only sequence that codes for
protein.
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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.
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Electrophoresis
Agarose Gel Electrophoresis separates DNA
fragments based on their size. DNA fragments are
often detected using fluorescence.
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Agarose gel electrophoresis can be used to
investigate an individuals 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.
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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.
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• 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.
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Southern Blotting
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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.
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Genome Sequencing Strategies
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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.
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Microarray Hybridization
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How was this experiment carried out? What are
the conclusions?
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Investigating the genotype of individuals can
answer questions about phylogeny (relatedness).
PCR
Liquify mite Purify DNA
Mt COI gene
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