what if we want to know what allele(s) of beta-globin an individual has?
TRANSCRIPT
What if we want to know what allele(s) of beta-globin an individual has?
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
33
3
3
3
3
55
5
55
5
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
33
3
3
3
3
55
5
55
5
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
DdeI cuts: CTNAG
Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Fig. 20-11TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II III
I II IIII II III
Radioactively labeledprobe for insulin gene
DNA + restriction enzyme
III HeterozygoteII mutant insulinallele
I Normalinsulinallele
Film overblot
Probe detectionHybridization with radioactive probe
Fragment frommutantInsulin allele
Fragments fromnormal insulinallele
Probe base-pairswith fragments
Nitrocellulose blot
1
4 5
32
Fig. 20-11TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II III
I II IIII II III
Radioactively labeledprobe for insulin gene
DNA + restriction enzyme
III HeterozygoteII mutant insulinallele
I Normalinsulinallele
Film overblot
Probe detectionHybridization with radioactive probe
Fragment frommutantInsulin allele
Fragments fromnormal insulinallele
Probe base-pairswith fragments
Nitrocellulose blot
1
4 5
32
Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Another option: PCR of Beta-globin gene, followed by DdeI digest
How can we measure gene expression?
vs.
wild type dif1
1. Isolate RNA2. Compare gene expression
Fig. 20-13
TECHNIQUE
RESULTS
Gel electrophoresis
cDNAs
-globingene
PCR amplification
Embryonic stages
Primers
1 2 3 4 5 6
mRNAscDNA synthesis 1
2
3
Reverse Transcriptase PCR (RT-PCR)
50 µm
Where in the organism is my gene transcribed? Promoter : reporter fusions
Fig. 20-14
50 µm
Where in the organism is my mRNA present? In situ hybridization
Fig. 20-15
TECHNIQUE
Isolate mRNA.
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresentingspecific genes
DNA microarraywith 2,400human genes
DNA microarray
1
2
3
4
WT
dif1
∆ dif1
myb98
∆ myb98
genes
Example of array data
TECHNIQUE
Gel electrophoresis
cDNAs
PCR amplificationPrimers
mRNAscDNA synthesis 1
2
3
Reverse Transcriptase PCR (RT-PCR)
Large scale sequencing of cDNA fragments
Sequence large numbers (millions) of cDNA fragments
No UV(3 samples)
UV(3 samples)
Large scale sequencing of cDNA fragments
Fragments matching rad51