southern blotting (sb) 4 jan 2015 final

SOUTHERN BLOTTING :

TECHNIQUE AND

APPLICATIONS

By Dr Ichha PurakUniversity ProfessorDepartment of BotanyRanchi Women’s College,Ranchi

Southern Blotting: Technique and Applications

101/04/15

SOUTHERN BLOTTING (HYBRIDIZATION )

Blotting techniques are used to transfer DNA or RNA fragments or

proteins from electrophoresis gel to a nitrocellulose sheet or nylon

membrane as blotting paper is used to blot ink.

Southern blotting is the transfer of DNA fragments from an

electrophoresis gel to a membranous support which results in

immobilization of DNA fragments. These immobilized single stranded

DNA fragments can then be subjected to hybridization with a labeled

probe.


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STEPS OF SOUTHERN BLOTTING01/04/15 3


Southern blotting was named after Edward M. Southern who developed

this procedure at Edinburgh University in the 1975.

It allows investigators to locate a particular sequence of DNA within a

complex mixture.

DNA (genomic or other source) is digested with a restriction enzyme and

separated by gel electrophoresis and transferred from an agarose gel onto

a Nitrocellulose sheet or Nylon membrane which is then incubated with a

single stranded DNA probe with known sequence. This probe is supposed

to form base pairs with its complementary DNA sequence and to form a

double-stranded DNA molecule.

The probe is labeled before hybridization either radioactively or is treated

enzymatically by alkaline phosphotase or horseradish peroxidase .

Finally, the location of hybridization with the probe is detected either by

directly exposing the membrane to X-ray film or by chemiluminescent

methodsSouthern Blotting: Technique and

Applications401/04/15

STEPS IN SOUTHERN/NORTHERN BLOTTING

wells

Southern Blotting: Technique and Applications 501/04/15

Sir Edwin Mellor Southern ,Fellow Royal Society and Trinity

won Albert Lasker Award (2005) in Clinical Medical Research

for the invention of the Southern Blot in 1975 when he was

working as Professor of Biochemistry at the University of

Edinburgh ,which is now a common molecular biology

procedure to identify DNA sequence


601/04/15

Other blotting methods that employ similar principles but using RNA or

Protein are named as Northern Blot and Western Blot in reference to

original Southern Blot

In Northern hybridization (Blot ) RNAs are transferred from gel to DBM

( Diazobenzyl oxy methyl) paper or Nylon membrane and are fixed by

baking. Denaturation step is not needed and the probe used for

hybridization is single stranded DNA Since the base-pairing is in a

sense the reverse of a "southern" experiment, this technique is referred

to as a "northern blot”.


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The Western blot (Protein immunoblot) is widely used analytical technique to

detect specific proteins in the tissue homogenate or extract. Proteins after

extraction are separated by gel electrophoresis based on length of polypeptide.

Proteins are transferred to a membrane e.g. Nitrocellulose or Poly vinyl di

fluoride (PVDF) and are probed by using specific antibodies specific to target

protein or antigen. The technique is also called immunoblotting and is to identify

antigens during infection or disease.

SOUTHERN BLOTTING

WESTERN BLOTTING01/04/15 8


FOR PERFORMING SOUTHERN BLOTTING FOLLOWING BROAD STEPS ARE TO BE TAKEN

• DNA ISOLATION AND PURIFICATION

• DIGESTION OF DNA BY RESTRICTION ENDONUCLEASES

• SEPARATE DNA FRAGMENTS BY GEL ELECTROPHOREIS

• DS STRANDED DNA FRAGMENTS ON GEL ARE DENATURED BY ALKALINE

TREATMENT TO GIVE SINGLE STRANDED DNA FRAGMENTS

BLOTTING (TRANSFER OF DNA FRAGMENTS FROM GEL TO NITROCELLULOSE

SHEET /NYLON MEMBRANE)

HYBRIDIZATION OF IMMOBILIZED DNA FRAGMENT WITH SINGLE STRANDED

RADIOACTIVELY LABELLED DNA PROBES

DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR

CHEMILUMNISCENT METHODS


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These steps shall be briefly discussed one by one•DNA ISOLATION AND PURIFICATION

For DNA isolation various protocols have been designed for different type

of sources and many rapidly working kits are available

•DNA extraction from Blood

•DNA extraction from serum

•DNA extraction from Plasmids

•DNA extraction from animal tissue

•DNA extraction from plant tissue

DNA isolation is a routine procedure to procure DNA for various

molecular techniques such as RDT, DNA sequencing,restriction

mapping ,forensic analysis , construction of genomic library etc.


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DNA extraction protocols involve following common steps

1.Cell disruption or cell lysis by physical methods as blending or

grinding .Lysis frees cellular proteins ,DNA & RNA

2. Removing membrane lipids by detergents

3. Removing proteins by protease and RNA by Rnase enzymes

4. DNA precipitation by alcoholsFinally by repeated centrifugation DNA is obtained in the form of pellet.


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For genomic isolation of DNA from plants Virginia Walbot and other similar

methods are employed. As plants are rich in proteins, polysaccarides

and lipids the protocol involves extraction of DNA using Tris chloride

buffer (pH-8), Na2EDTA, Sodium Chloride and Sodium Dodicyl Sulphate

(SDS). Precipitation of DNA is done by Ammonium Acetate and Ethanol

.The extracted DNA by this method can be stored for long duration. A part

of extracted DNA as pellet can be subjected to confirmation by using

Diphenylamine (DPA) reagent.

For extraction of DNA from blood many protocols are in practice . Some protocols are quite rapid taking only 15-30 minutes employing simple lysis with proteinase K enzyme. Protocol for extraction of genomic DNA from whole blood Reagents

Buffer A (Red blood cell lysis buffer) composition

•0.32 M sucrose

•10 mM Tris HCl

•5 mM MgCl2

•0.75% Triton-X-100 Adjust pH to 7.6

Buffer B (Proteinase K buffer) composition

•20 mM Tris-HCl

•4 mM Na2EDTA

•100 mM NaCl Adjust pH to 7.4

All solutions should be sterile. Buffer A should be autoclaved prior to

addition of Triton-X-100. Sterile filtering of solutions instead of autoclaving

is a better option. Southern Blotting: Technique and Applications

1201/04/15

Procedure1.2 ml of buffer A is added to 2 ml of blood and 4 ml of cold, sterile,

distilled, deionised water. The mixture is poured in centrifuge tubes.

Tubes are spinned gently and left on ice for 2-3 minutes for incubation.

2. Tubes are further spinned for 15 minutes at 3500 rpm at 4oC.

Supernatant is discarded and pellet is resuspended in 2ml of buffer A

and 6 ml of water. The tubes are again spinned for 15 minutes at 3500

rpm at 4oC. The pellet should be white or cream in colour if red it is

washed again.

3. 5ml of Buffer B and 500 µl of 10% SDS is added to the pellet and

spinned for 30-60 seconds. Then 50 µl of freshly prepared Proteinase

K solution (20mg/ml) is added.


1301/04/15

4. Left for incubation for two hours at 55oC in a water bath. After that

left to cool at room temperature. Then 4 ml of 5.3 M NaCl solution is

added and spinned at 4500 rpm for 15-20 minutes at 4oC.

5. Supernatant is poured off into a fresh tube .Care should be taken

not to dislodge pellet. Equal volume of isopropyle alcohol stored at

20oC is added and tubes are shaked 5-6 times to precipitate DNA.

6. Pellet is transferred to microfuge tube and washed with 1 ml of

70% ethanol. DNA is left to dry for 15-20 minutes at 37oC. DNA is

resuspended in 300-400 µl of Tris HCL (ph 8.5). Left overnight to

dissolve at room temperature and can be refrigerated and stored for

up to a year in ethanol.


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•DIGESTION OF DNA BY RESTRICTION ENDONUCLEASE

Next step is digesting DNA into several pieces of different sizes. This is

done using one or more restriction endonucleases.

Restriction enzymes are isolated from bacteria that recognize specific

sequences in DNA and then cut the DNA to produce fragments, called

restriction fragments or RFLP (Restriction Fragment Length

Polymorphism ) with either blunt or cohesive ends. A unique feature of

these enzymes is that they cleave the DNA at palindromic sequences

that are 4-8 base pairs in length. Thus the enzyme EcoRI cleaves the

DNA wherever the sequence,

Is found. Now more than 60 of these enzymes are known, and by a

suitable choice of restriction enzymes, the DNA can be cut at

appropriate positions .

.


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•SEPARATION OF DNA FRAGMENTS BY GEL

ELECTROPHOREIS

Electrophoresis is a common lab technique used to identify, quantify, and

purify nucleic acid fragments. DNA samples cut by restriction

endonuclease are loaded into wells of an agarose or acrylamide gel and

subjected to an electric field, causing the negatively charged nucleic

acids to move toward the positive electrode. Shorter DNA fragments will

travel more rapidly, whereas the longest fragments will remain closest to

the origin of the gel (near well) , resulting in separation of DNA

fragments based on size.


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An Agarose Gel after electrophoresis showing DNA fragments separated by size


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Agarose remains the most widely used gel matrix for separating nucleic

acid fragments because it is nontoxic, easy to use, and offers a broad

separation range. Varying the agarose concentration controls the gel

pore size, facilitating the separation of a wide range of different-size

nucleic acids. The migration of nucleic acids in agarose gels is also

affected by the choice of buffer and applied voltage

Polyacrylamide is a cross-linked polymer that provides very high

resolution of DNA molecules in the 10–3,000 bp size range. Under the

appropriate conditions, DNA molecules differing in size by a single base

pair can be resolved

DNA is loaded by micropipette in the wells casted on the gel plate with

the help of comb. Ethidium bromide is added to DNA sample before

loading or in the electrophoretic buffer.


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Fragmented DNA is typically electrohoresed on an agarose gel to

separate the fragments according to their molecular weights.

Acrylamide gels can alternatively be used for good resolution of smaller

DNA fragments (<800 bp).

Agarose gel electrophoresis is ideal for rapid, high-resolution

electrophoresis of restriction digests, PCR reactions.

Because there are so many different restriction fragments on the gel, it

usually appears as a smear rather than discrete bands.

For determination of DNA size, a wide range of DNA ladders are

available for accurate size and mass estimations, including 100 bp

ladders and 1 Kb Plus ladders which can be loaded in 1-2 wells.

Gel loading buffers include one or two tracking dyes as bromophenol

blue or xylene cyanol to monitor the progress of electrophoresis by

migration of the dye


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DS STRANDED DNA FRAGMENTS ON GEL ARE

DENATURED BY ALKALINE TREATMENT TO GIVE SINGLE

STRANDED DNA FRAGMENTS

The DNA is denatured into single strands by incubation with 0.5 M

NaOH so as to obtain single stranded DNA . These negatively charged

SS DNA can bind to positively charged membrane. This step also

destroys any residual RNA that may still be present with the DNA or

denaturation may be caused by high temperature treatment.


2001/04/15

A depurination step is optional. Fragments greater than 15 kb are hard to

transfer to the blotting membrane. Keeping this in view the gel may be

treated with dilute HCL for about 15 minutes which can depurinate DNA

fragments ,breaking the DNA into smaller fragments.

A neutralization step by Nacl is required after alkali or acid treatment to

prevent base pairng before hybridization with probe

BLOTTING (TRANSFER OF DNA FRAGMENTS FROM GEL TO

NITROCELLULOSE SHEET /NYLON MEMBRANE)

To transfer denatured single stranded DNA from gel to a solid

support a sheet of nitrocellulose or nylon membrane is placed on the

top of the gel.

Above the gel stacks of paper towels are placed . A glass can be placed on

the paper towels on which a weight is placed to ensure good and even

contact between gel and membrane. A wick of whatman filter paper is used

for buffer transfer from a region of high water potential to a region of low

water potential by inducing capillary action. In place of paper wick sponge

sheet can also be used

With this buffer transfer DNA strands are transferred from gel to membrane.

Ion exchange interaction binds the DNA to the membrane due to negative

charge of DNA and positive charge of membrane.Southern Blotting: Technique and

Applications2101/04/15

Many scientists feel nylon is better since it is less fragile.

Transfer is usually done by capillary action, which takes several hours.

Capillary action transfer draws the buffer up through the gel onto the

membrane, ssDNA fragments are drawn along with buffer.

A vacuum blot apparatus can be used instead of capillary action. In this

procedure, a vacuum sucks SSDNAs through the membrane.

The membrane is then baked at about 80ᵒ C for about two hours to

permanently attach the transferred DNA to the membrane or the

membrane is exposed to ultraviolet radiation.

Care should be taken while baking the Nitrocellulose sheet at high

temperature as it is highly cumbustible


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TRANSFER OF FRAGMENTS FROM GEL TO MEMBRANE


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BLOTTING ARRANEMENT

Weight

HYBRIDIZATION OF IMMOBILIZED DNA FRAGMENT

WITH SINGLE STRANDED DNA PROBES.

For hybridization a nucleic acid probe ( DNA or RNA ) with sequence

homologous to the target sequence is labeled with radioactivity,

fluorescent dye, or an enzyme that can generate a chemiluminescent

signal when incubated with the appropriate substrate. The choice of

the label depends on several factors such as the nature of probe or

probe template, sensitivity needed, quantification requirements, ease

of use, and experimental time. For this the Nitrocellulose sheet bearing

ssDNA fragments is placed in a bag containing a solution of

radioactively labeled single stranded DNA probe with known

sequence.


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32 P ATP is used to label the probe radioactively. After hybridization,

excess or unhybrized probe is washed from the membrane in

several changes of buffer The wash can be with low or high

stringency which can remove hybridization solution and also unused

probes

The result is that only fully hybridized labeled probe molecules, with

complementary sequence to the region of interest, remain bound.

A pre-hybrdization step is required before hybridization to block

non-specific sites, to prevent single-stranded probe binding just

anywhere on the membrane.


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DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR CHEMILUMNISCENT METHODS

In the detection step, the bound, labeled probe is detected using the

method required for the particular label used. For example, radiolabeled

probes may be detected using X-ray film or a phosphorescense imaging

instrument, and enzymatically labeled probes are typicallly detected by

incubating with a chemiluminescent substrate and exposing the blot to

X-ray film.

.

The transfer step of the DNA from the electrophoresis gel to a

membrane permits easy binding of the labeled hybridization probe to the

size-fractionated DNA. It also allows for the fixation of the target-probe

hybrids, required for analysis by autoradiography or other detection

methods.


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In order to analyze a Southern Blot, a radioactive genetic probe is used

in a hybridization reaction with the DNA . X-ray is taken of the Southern

Blot after a radioactive probe has been allowed to bind with the

denatured DNA on the paper, only the areas where the radioactive probe

binds will show up on the film. This allows researchers to identify, in a

particular person's DNA, the occurrence and frequency of the particular

genetic pattern contained in the probe.

Hybridization of the probe to a specific DNA fragment on the filter

membrane indicates that this fragment contains DNA sequence that is

complementary to the probe


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ab

d c

a) DNA after digestion by RE ,Fragments separated on Agarose Gelb) Denatured DNA fragments are transferred from Gel to Nitrocellulose Sheet (Blotting)c) The Nitrocellulose sheet is incubated with specific single stranded DNA probed) The location of the DNA fragment that hybridizes with the probe can be displayed by

autoradiography.


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The Southern blot is used to detect the presence of a particular bit of DNA in a sample

Extracted and purified DNA

DNA digested with restriction endonuclease Restriction fragments

DNA is loaded into wells of the gel

DNA fragments move on gel as per size

SSDNA fragments transferred to NC sheet

Development of Blot

denaturationElectric currentDNA probe added


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01/04/15Southern Blotting: Technique and

Applications32

APPLICATIONS OF SOUTHERN BLOTTING

Suthern Blotting is a very useful techniquae It can be used for identification of DNA fragment in a DNA sample

It is significantly useful in identification of criminals and is a tool in

forensics

Diagnosis of Infectious disease and HIV-1

Used in personal Identification

Used in phyllogenetic analysis

Used in paternity and maternity tests

It can be used to map the restriction sites and make RFLP maps.

Used to identify mutations,deletions and gene rearrangements.

Used in prenatal diagnosis of genetic diseases (Sickle cell anaemia)

Southern blots performed with restriction enzyme-digested

genomic DNA may be used to determine the number of sequences

(e.g., gene copies) in a genome .

It is used for detection and identification of the transferred genes

in transgenic individuals.

In regards to genetically modified organisms, Southern blotting is

used as a definitive test to ensure that a particular section of DNA

of known genetic sequence has been successfully incorporated

into the genome of the host organism

It can be used to map the restriction sites and make RFLP maps.

Southern Blotting can also be used to follow the inheritance of

selected genes

Southern Blotting and DNA fingerprinting

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Mutations within restriction sites change the sizes of restriction

fragments and as a result, the positions of bands in the Southern-blotting

analysis and autoradiography also change.

This change in position can later be compared to normal blot-analyses in

order to reveal where the possible change has occurred. The existence of

genetic diversity created by these mutations in a population, is termed

polymorphism.

The detected mutation in turn may have different effects. It may cause

disease . Some examples of such diseases include sickle-cell anemia,

cystic fibrosis, and Huntington chorea.

Polymorphism refers to the DNA sequence variation between

individuals of a species. If the sequence variation occurs at the restriction

sites, it could result in RFLP. The most well known example is the RFLP

due to globin gene mutation. β

Southern Blotting and DNA fingerprinting

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All of these mutations can be detected by comparing the restriction-

fragment-length polymorphisms with normal fragments of DNA .

It is also used to determine the molecular weight of a restriction

fragment and to measure relative amounts in different samples. Under

optimal conditions, Southern blotting detects ~ 0.1 pg of the DNA of

interest

.

Detection of the sickle-cell globin gene by Southern blotting. The base change (A T) that causes sickle cell anaemia a → MstII target site that is present in the normal -globin gene. This difference can be detected by βSouthern blotting.

Use of Southern Blotting to detect sickle cell globin gene

RFLP resulting from -globin gene mutation. β

In the normal cell, the sequence corresponding to 5th to 7th amino acids of

the -globin peptide is CCTGAGGAG, which can be recognized by the β

restriction enzyme MstII.

In the sickle cell, one base is mutated from A to T, making the site

unrecognizable byMstII. Thus, MstII will generate 0.2 kb and 1.2 kb

fragments in the normal cell, but generate 1.4 kb fragment in the sickle

cell. These different fragments can be detected by the southern blotting

SOUTHERN BLOTTING (A-E)

END OF PRESENTATION THANKS

01/04/15 38Southern Blotting: Technique and

Applications

southern blotting (sb) 4 jan 2015 final

Science

dna sequence southern

blotting paper

ranchisouthern blotting

southern experiment

analytical technique

dna genomic

single stranded dna

transfer of dna fragments