Northern, Southern, Western

Ed Southern:

In the 1970s Ed Southern of

Oxford University invented a

revolutionary DNA blotting

technique.

The Southern Blot allows the

visualization of one DNA

fragment from a whole

genome DNA extract.

Northern and Western

• People then applied the same technique

to RNA.

• They called it a “Northern blot”.

• Then other people applied it to protein,

and imaginatively called it a “Western

blot” Funny, eh?

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Introduction Concept: reannealing nucleic acids to identify sequence of interest.

Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.

Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.

Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.

Size bands and quantify abundance of molecule.

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Southern Blot: DNA-DNA*

Developed by Edwin Southern.

Uses gel electrophoresis together with hybridization

probes to characterize restriction fragments of

genomic DNA (or DNA from other sources, such as

plasmids).

Identifies DNA with a specific base sequence.

Can be done to detect specific genes present in cells.

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Southern Steps

1. DNA to be analyzed is digested to completion with a

restriction endonuclease.

2. Electrophoresis to maximally separate restriction

fragments in the expected size range. A set of

standards of known size is run in one lane of the gel.

3. Blot fragments onto a nitrocellulose membrane.

4. Hybridize with the 32P probe.

5. Autoradiography.

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Step 2

Gel electrophoresis

• Separates DNA fragments.

Soak gel in 0.5 M NaOH

• Converts dsDNA to ssDNA

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Step 3. Nitrocellulose Blot • Cover gel with nitrocellulose

paper…then…

• Cover nitrocellulose paper with thick layer of paper towels.

• Compress apparatus with heavy weight.

• ssDNA binds to nitrocellulose at same position it had on the gel.

• Vacum dry nitrocellulose at 80C to permanently fix DNA in place or cross link (via covalent bonds) the DNA to the membrane.

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Step 4. Hybridization • Incubate nitrocellulose sheet with

a minimal quantity of solution

containing 32P-labeled ssDNA

probe.

• Probe sequence is complementary

to the DNA of interest.

• Incubate for several hours at

suitable renaturation temperature

that will permit probe to anneal to

its target sequence(s).

• Wash & dry nitrocellulose sheet.

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Step 5. Autoradiography

• Place nitrocellulose sheet over

X-ray film.

• X-ray film darkens where the

fragments are complementary

to the radioactive probes.

Characterization: Southern blot hybridization

-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)

-developed by Edwin Southern

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Southern Application: Diagnosis & detection of genetic diseases.

• Used to diagnose sickle cell-anemia.

• AT base change in the subunit of Hb

Glu Val.

• Development of a 19 residue oligonucleotide probe

complementary to sickle-cell gene’s mutated segment.

• Probe hybridizes to DNA from homozygotes of sickle-cell

anemia but not from normal individuals.

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Northern Blot: RNA-

DNA*(RNA*)

• Alwine adapted Southern's method for DNA to detect, size and quantify RNA – 1977.

• No need to digest RNA with restriction enzymes.

• Use formaldehyde to break H-bonds and denature RNA because single-stranded RNA will form intramolecular base pairs and "fold" on itself.

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Northern Steps

1. Isolate RNA & treat with formaldehyde.

2. Electrophorese RNA in denaturing agarose gel (has

formaldehyde). Visualize RNA in gel using Ethidium

bromide stain and photograph.

3. Transfer single-stranded RNA to nitrocellulose or nylon

membrane. Covalently link RNA to membrane.

4. Incubate membrane (RNA immobilized on membrane) with

labeled DNA or RNA probe with target sequence.

5. Development.

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Step 1 Isolate RNA:

-To detect rare mRNA, isolate the poly A+ mRNA.

-RNA is both biologically and chemically more labile than

DNA. Thus eliminate RNases.

Step 2

Electrophoresis:

- Performed in formaldehyde agarose gel to prevent RNA from

folding on itself.

- Stain with EtBr to visualize the RNA bands.

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Step 3 -Transfer single-stranded RNA to nitrocellulose or nylon

membrane:

Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used.

Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile.

-Covalently link RNA to membrane:

UV cross linking is more effective in binding RNA to the membrane than baking at 80C.

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Step 4 & 5 -Prehybridize before hybridization:

Blocks non-specific sites to prevent the single-stranded probe from binding just anywhere on the membrane.

-Incubate membrane with labeled DNA or RNA probe with target sequence:

Probe could be 32P, biotin/streptavidin or a bioluminescent probe.

-Autoradiography:

Place membrane over X-ray film.

X-ray film darkens where the fragments are complementary to the radioactive probes.

Ethidium bromide is fluorescent in

UV light

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Northern Application

• Northern blots are particularly useful for determining the conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.

• For instance:

When trying to learn about the function of a certain protein, it is sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a cDNA clone of the protein of interest as the probe.

Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe.

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Summary

Southern

• DNA on membrane.

• Digest DNA.

• Convert dsDNA to

ssDNA.

• Probe with DNA or

RNA.

Northern

• RNA on membrane.

• No need to digest DNA.

• Denature “folded” RNA

with formaldehyde.

• Probe with DNA or RNA.

Characterization: Western blotting

-transfer of protein from a gel to a membrane (e.g., nitrocellulose,

nylon)

-requires the use of an electric current to facilitate transfer

X Protein

X

x Buffer; requires electric current

X

React with

Antibody

X

Enzyme

reaction

or

Fluorescence in situ

Hybridization

Fluorescence in situ

Hybridization (FISH)

• FISH - a process which vividly paints

chromosomes or portions of chromosomes

with fluorescent molecules. Identifies

chromosomal abnormalities

• Aids in gene mapping, toxicological studies,

analysis of chromosome structural

aberrations, and ploidy determination

FISH

• Used to identify the presence and location

of a region of DNA or RNA within

morphologically preserved chromosome

preparations, fixed cells or tissue sections.

• This means you can view a segment or

entire chromosome with your own eyes

• Was often used during M phase but is now

used on I phase chromosomes as well

FISH

• Advantage: less labor-intensive

method for confirming the

presence of a DNA segment

within an entire genome than

other conventional methods like

Southern blotting.

FISH Procedure

• Denature the chromosomes

• Denature the probe

• Hybridization

• Fluorescence staining

• Examine slides or store in the

dark.

FISH Procedure

Probes

Biotin

Complementary sequences of

target nucleic acids

• Designed against the sequence

of interest

• Probes are tagged with

fluorescent dyes like biotin,

fluorescein, Digoxigenin

• Size ranges from 20-40 bp to

1000bp

Fluorescein

FISH Uses

• Detection of high concentrations of

base pairs

• Eg: Mouse metaphase preparation

stained with DAPI (4',6-diamidino-2-

phenylindole) is a fluorescent stain that binds

strongly to A-T rich regions in DNA. It is

used extensively in fluorescence microscopy.

FISH Uses Centromere regions stained brighter - means they are

rich in A-T bonds

Also used in germ cell or prenatal diagnosis of

conditions such as aneuploidies (abnormal number of

chromosomes in a cell).

FISH and Telomeres • Telomeric probes define the terminal boundaries of

chromosomes (5’ and 3’ ends)

• Used in research of chromosomal rearrangements

and deletions related to cell aging or other genetic

abnormalities.

• Special telomeric probes specific to individual

chromosomes have been designed.

• Probe is based on the TTAGGG repeat present on all

human telomeres.

FISH and Telomeres

Application in

cytogenetics - can detect

submicroscopic deletions

and cryptic translocations

of genes associated with

unexplained mental

retardation and

miscarriages

FISH - Medical

FISH can be used in the

study of transgenic

animals (eg: Polly)

Selective markers show

if the human DNA was

inserted successfully

and pinpoint where the

human DNA is .

Diagnostic Applications of FISH • Prenatal diagnosis

• Cancer diagnosis

• Molecular cytogenetic of birth defects and mental

retardation

• The identification of specific chromosome

abnormalities

• The characterization of marker chromosomes

• Interphase FISH for specific abnormalities in cases of

failed

• Cytogenetic

• Monitoring the success of bone marrow transplantation

Other Human Disorders

• Sickle Cell Anemia –

– autosomal recessive

– Found most often among people of African

ancestry

– Blood cells sickle (change shape) when

oxygen-deprived (exertion, increase in altitude)

– Causes sickle cell event – pain and immobility

and death of tissue ( dangerous if in organ)

– Treatment – hospitalization and oxygen

– Carriers are resistant to malaria

Tay Sachs Disease

• Autosomal recessive ( Gangliosidosis or

Hexosaminidase A deficiency) .

• Genetic mutation of HEXA gene on

chromosome 15

• Found most often among Jews of Mediterranean

ancestry

• Child born appearing normal, but fat builds up in

brain and child dies by age 5

• No treatment, no cure

Huntington Disease

• It is a neurodegenerative genetic disorder that

affects muscle coordination and leads to

cognitive decline and dementia

• Autosomal dominant

– Symptoms do not appear until age 30-40.

– Death takes about 5-10 years

– No treatment, no cure – but there is a test to see if

you have it before symptoms begin

– Results in mental impairment and uncontrollable

spastic movements

Deletions • Chromosome fragment breaks off and is lost

• Cri du chat syndrome – mental retardation and

cat-like cry, delayed development, distinctive

facial features, small head size (microcephaly),

widely-spaced eyes (hypertelorism), low birth

weight and weak muscle tone (hypotonia) in

infancy.

• It is a rare genetic condition that is caused by the

deletion (a missing piece) of genetic material on

the small arm (the p arm) of chromosome 5.

The condition affects an estimated 1 in 20,000 to

50,000 live births, strikes all ethnicities, and is more

common in females by a 4:3 ratio

Prenatal Tests to detect

chromosomal problems:

• Amniocentesis – removes a little amniotic fluid

from around baby – fluid is then tested for

abnormal proteins and the cell in it can be

karyotyped.

• Amniocentesis usually done between 16 to 20

weeks, which is during second trimester.

• Risk of miscarriage

Chorionic Villus Sampling

• Take a piece of the chorionic villus from the

placenta – it is made of baby cells – and test

as in amniocentesis It is done during

early pregnancy, most often between the

10th and 12th weeks.

– Risk of miscarriage

– Has been linked to deformed fingers

Bioethical Dilemma

• Once a prenatal diagnosis of a genetic disorder

is made, what are the parents going to do?

– Do nothing and give birth to child with disorder

– Abort embryo/fetus

• Who should make the decision?

• What should enter into making the decision?

Genetic Counseling

• Genetic counselor:

– educates the parents about the disorder,

– tells them of their options without

influencing their decision,

– and tells them of the consequences of

each option .

DNA Fingerprints

• The pattern of DNA formed during gel

electrophoresis. Used by law enforcement.

“DNA in the Courtroom”

1. Use of VNTRs (variable number of tandem

repeats; different individuals have different

numbers of repetitive stretches of DNA, for

example, GGAGG). One individual might

have 6, another 12.

2. VNTRs can be analyzed by gel

electrophoresis, creating a banding

pattern specific to each individual—like a

bar code .

END

Part II