Transcript cDNA libraries, Microarray Analysis

Additional Powerful Molecular Techniques
Synthesis of cDNA (complimentary DNA)
Polymerase Chain Reaction (PCR)
Microarray analysis
Link to Gene Therapy information (in syllabus)
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!
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
LE 20-14
Tissue sample
Isolate mRNA.
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of singlestranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.
Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)
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 cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
LE 20-7
5
3
Target
sequence
Genomic DNA
Denaturation:
Heat briefly
to separate DNA
strands
3
5
5
3
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3
Cycle 1
yields
2
molecules
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
primer
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
5
Primers
New
nucleotides
Gene Therapy
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