What if we want to know what allele(s) of beta-globin an individual has?

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