Additional Powerful Molecular Techniques

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Additional Powerful Molecular Techniques
Synthesis of cDNA (complimentary DNA)
Polymerase Chain Reaction (PCR)
Microarray analysis
Link to Gene Therapy information (in syllabus)
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cDNA a DNA compliment of RNA template
Why DNA is more stable than RNA and DNA copies
of RNA can be cloned into vectors
-First strand synthesis uses RNA as a template
to make a complimentary single stranded
DNA -Second strand synthesis uses first DNA
strand as a template to make double stranded
cDNA for cloning
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What is the source of the RNA?
Often mRNA
What is a major difference between prokaryotic
eukaryotic genes?
Eukaryotic genes have introns that interrupt the
protein coding sequence.
If you were interested in eukaryotic protein
coding sequences, why would it be advantageous to
use cDNA clones or libraries rather than genomic
clones or libraries?
Since cDNA is made from mature mRNA, no introns!
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Studying Expression of Interacting Groups of Genes

• Automation has allowed scientists to measure
  expression of thousands of genes at one time
  using DNA microarray assays
• DNA microarray assays compare patterns of gene
  expression in different tissues, at different
  times, or under different conditions

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LE 20-14
Tissue sample
Isolate mRNA.
mRNA molecules
Make cDNA by reverse transcription, using
fluorescently labeled nucleotides.
Apply the cDNA mixture to a microarray, a
microscope slide on which copies of
single-stranded DNA fragments from the organisms
genes are fixed, a different gene in each spot.
The cDNA hybridizes with any complementary DNA on
the microarray.
Labeled cDNA molecules (single strands)
DNA microarray
Rinse off excess cDNA scan microarray for
fluorescent. Each fluorescent spot (yellow)
represents a gene expressed in the tissue sample.
Size of an actual DNA microarray with all the
genes of yeast (6,400 spots)
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Amplifying DNA in Vitro The Polymerase Chain
Reaction (PCR)

• The polymerase chain reaction, PCR, can produce
  many copies of a specific target segment of DNA
• A three-step cycleheating, cooling, and
  replicationbrings about a chain reaction that
  produces an exponentially growing population of
  identical DNA molecules

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LE 20-7
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3
Target sequence
Genomic DNA
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3
Denaturation Heat briefly to separate DNA strands
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Annealing Cool to allow primers to form hydrogen
bonds with ends of target sequence
Cycle 1 yields 2 molecules
Primers
Extension DNA polymerase adds nucleotides to the
3 end of each primer
New nucleo- tides
Cycle 2 yields 4 molecules
Cycle 3 yields 8 molecules 2 molecules (in white
boxes) match target sequence
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Gene Therapy
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