Download Measuring expression - May 18

What if we want to know what allele(s) of beta-globin an individual has?
Wild-type hemoglobin DNA
C T T
3
5
G A A
Mutant hemoglobin DNA
C A T
5 3
G T A
3 5
mRNA
5
5
3
mRNA
G A A
Normal hemoglobin
Glu
3 5
G U A
Sickle-cell hemoglobin
Val
3
Wild-type hemoglobin DNA
C T T
3
5
G A A
Mutant hemoglobin DNA
C A T
5 3
G T A
3 5
mRNA
5
5
3
mRNA
G A A
Normal hemoglobin
Glu
DdeI cuts: CTNAG
3 5
G U A
Sickle-cell hemoglobin
Val
3
Fig. 20-10
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
DdeI
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
Fig. 20-11
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal
insulin
allele
II mutant
insulin
allele
III Heterozygote
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
2 Gel electrophoresis
3 DNA transfer (blotting)
Radioactively labeled
probe for insulin gene
I
II III
Probe base-pairs
with fragments
Fragment from
mutant
Insulin allele
Nitrocellulose blot
4 Hybridization with radioactive probe
Fragments from
normal insulin
allele
I
II III
Film
over
blot
5 Probe detection
Fig. 20-11
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal
insulin
allele
II mutant
insulin
allele
III Heterozygote
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
2 Gel electrophoresis
3 DNA transfer (blotting)
Radioactively labeled
probe for insulin gene
I
II III
Probe base-pairs
with fragments
Fragment from
mutant
Insulin allele
Nitrocellulose blot
4 Hybridization with radioactive probe
Fragments from
normal insulin
allele
I
II III
Film
over
blot
5 Probe detection
Fig. 20-10
Another option: PCR of Beta-globin gene,
followed by DdeI digest
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
DdeI
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
How can we measure gene expression?
wild type
dif1
vs.
1.
2.
Isolate RNA
Compare gene expression
Fig. 20-13
TECHNIQUE
1 cDNA synthesis
Reverse Transcriptase PCR (RT-PCR)
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
1 2 3 4 5 6
Where in the organism is my gene transcribed?
Promoter : reporter fusions
50 µm
Fig. 20-14
Where in the organism is my mRNA present?
In situ hybridization
50 µm
Fig. 20-15
TECHNIQUE
1 Isolate mRNA.
2 Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
3 Apply the cDNA mixture to a
microarray, a different gene in
each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA fragments
representing
specific genes
DNA microarray
4 Rinse off excess cDNA; scan
microarray for fluorescence.
Each fluorescent spot represents a
gene expressed in the tissue sample.
DNA microarray
with 2,400
human genes
Example of array data
genes
WT
dif1
∆ dif1
myb98
∆ myb98
Large scale sequencing of cDNA fragments
TECHNIQUE
1 cDNA synthesis
Reverse Transcriptase PCR (RT-PCR)
mRNAs
cDNAs
2 PCR amplification
Primers
Sequence large numbers (millions) of cDNA fragments
3 Gel electrophoresis
Large scale sequencing of cDNA fragments
No UV
(3 samples)
UV
(3 samples)
Fragments matching rad51