DNA Technology- Cloning, Libraries, and PCR 12 and 15 November, 2004 Text Chapter 20 - PowerPoint PPT Presentation

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DNA Technology- Cloning, Libraries, and PCR 12 and 15 November, 2004 Text Chapter 20

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DNA can be cloned into bacterial plasmids for research or commercial applications. ... A library is a set of clones that carry different fragments, representing the ... – PowerPoint PPT presentation

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Title: DNA Technology- Cloning, Libraries, and PCR 12 and 15 November, 2004 Text Chapter 20


1
DNA Technology- Cloning, Libraries, and PCR 12
and 15 November, 2004 Text Chapter 20
2
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.
3
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.
4
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.
5
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.
6
A cDNA contains only sequence that codes for
protein.
7
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.
8
Electrophoresis
Agarose Gel Electrophoresis separates DNA
fragments based on their size. DNA fragments are
often detected using fluorescence.
9
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.
10
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.
11
  • 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.
12
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.
13
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.
14
How was this experiment carried out? What are
the conclusions?
15
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.
16
Genome Sequencing Strategies
17
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.
18
Microarray Hybridization
19
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20
Investigating the genotype of individuals can
answer questions about phylogeny (relatedness).
PCR
ITS regions (highly variable)
Liquify mite Purify DNA
21
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