SOUTHERN SOUTHERN BLOTTINGBLOTTING

M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

OUTLINEOUTLINEDNA SPECIMEN COLLECTION AND

STORAGEPROCEDUREWATCHPOINTS USES

DNADNAEach individuals unique genetic

blueprint is stored in material known as DNA.

DNA is found in all cells containing a nucleus.

DNA can be extracted for analysis from hair, bones, saliva, sperm, skin, organs, all body tissues and blood.

DNADNAThe deoxyribonucleic acid, DNA,

is a long chain of nucleotides which consist of:

1. Deoxyribose(sugar with 5 carbons)

2. Phosphate groups3. Organic(nitrogenous)bases

Nitrogenous BasesNitrogenous BasesTwo classes:Purines

◦Adenine◦Guanine

Pyrimidines◦Cytosine◦Thymine

DNADNADNA molecules are arranged in a

double helix which resembles a tightly coiled twisted ladder.

The sides of the ladder have alternating units of phosphate and deoxyribose sugar.

DNADNAThe rungs of the ladder are

formed by the nitrogenous “base pairs”.

Hydrogen bonds hold the strands together.

The bases bind together in a complementary fashion.

DNADNA

The base adenine (A) always pairs with thymine (T).

The base guanine (G) always pairs with cytosine (C).

DNADNAExampleFirst strand GGGTTTAAACCCSecond strand CCCAAATTTGGG

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

I. Temperature Storage for DNA◦Purified DNA may be refrigerated at 4°C for up to 3 years.

◦Samples kept over 3 years should be frozen at -70°C.

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

II. Specimens used in DNA testing◦Whole blood◦Solid tissue◦Serum and plasma◦Urine◦Bone marrow◦and many others

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

III. Specimen Collection Requirements◦A. Blood and Bone Marrow Collection tubes are EDTA or ACD 5-15 ml Samples should not be frozen for

transport 4-25°C

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

B. Serum◦Collection tubes with no additives

◦100 µl to 1 ml◦Transported at 20-25°C

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

Spin the samples to separate the plasma, RBC, and buffy coat.

Extract the buffy coatThe buffy coat is used because

the WBC are nucleated and contain DNA.

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

C. Tissue◦A sterile container with no formalin or paraffin must be used for collection.

◦30 mg ◦Dry ice should be used for transport.

DNA STORAGE AND DNA STORAGE AND COLLECTIONCOLLECTION

D. Urine◦Urine container should be used for collection.

◦At least 1 ml should be collected.

◦Transported at 4-25°C

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThe technique was developed by

E.M. Southern in 1975.The Southern blot is used to

detect the presence of a particular piece of DNA in a sample.

The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThe key to this method is

hybridization.Hybridization-process of forming

a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThere are 2 important features of

hybridization:◦The reactions are specific-the probes

will only bind to targets with a complementary sequence.

◦The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

SOUTHERN BLOTTINGSOUTHERN BLOTTING

SOUTHERN BLOTTINGSOUTHERN BLOTTINGSteps for hybridization

◦ 1. The mixture of molecules is separated.◦ 2. The molecules are immobilized on a

matrix.◦ 3. The probe is added to the matrix to bind

to the molecules.◦ 4. Any unbound probes are then removed.◦ 5. The place where the probe is connected

corresponds to the location of the immobilized target molecule.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGI. DNA Purification

◦Isolate the DNA in question from the rest of the cellular material in the nucleus.

◦Incubate specimen with detergent to promote cell lysis.

◦Lysis frees cellular proteins and DNA.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGProteins are enzymatically

degraded by incubation with proteinase.

Organic or non-inorganic extraction removes proteins.

DNA is purified from solution by alcohol precipitation.

Visible DNA fibers are removed and suspended in buffer.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGII. DNA Fragmentation

◦Cut the DNA into different sized pieces.

◦Use restriction endonucleases (RE)

◦Bacterial proteins◦In vivo, they are involved in DNA metabolism and repair or in bacterial host defense.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGNucleases hydrolyze the bonds that connect bases within the strand, resulting in cleavage of the strand.

They cleave the double stranded nucleic acid only at specific points.

SOUTHERN BLOTTINGSOUTHERN BLOTTING

This allows for specific sequences to be identified more readily.

Fragments are now easily separated by gel electrophoresis.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGIII. Gel Electrophoresis

◦Sorts the DNA pieces by size◦Gels are solid with microscopic pores

◦Agarose or polyacrimide◦Gel is soaked in a buffer which controls the size of the pores

◦Standards should also be run

SOUTHERN BLOTTINGSOUTHERN BLOTTING

Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGGels can be stained with

ethidium bromide. This causes DNA to fluoresce

under UV light which permits photography of the gel.

You can tell the exact migration of DNA standards and the quality of the RE digestion of the test DNA.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGHigh quality intact DNA should

give the appearance of a single band.

Degraded material will smear downwards.

Only a small amount of degradation is tolerable.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGIV. Blotting

◦Transfer the DNA from the gel to a solid support.

◦The blot is usually done on a sheet of nitrocellulose paper or nylon.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGDNA is partially depurinated with

dilute HCL which promotes higher efficiency transfer by breaking down fragments into smaller pieces.

DNA is then denatured with an alkaline solution such as NAOH.

This causes the double stranded to become single-stranded.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGDNA is then neutralized with NaCl

to prevent re-hybridization before adding the probe.

Transferred by either electrophoresis or capillary blotting.

SOUTHERN BLOTTINGSOUTHERN BLOTTING1) Electrophoresis- takes advantage of

the molecules negative charge.

SOUTHERN BLOTTINGSOUTHERN BLOTTING2) Capillary blotting-fragments are eluted from

the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThe blot is made permanent by:◦Drying at ~80°C◦Exposing to UV irradiation

SOUTHERN BLOTTINGSOUTHERN BLOTTINGV. Blocking

◦Buffer binds to areas on the blot not occupied by patient DNA.

◦Blocks the empty sites from being bound during hybridization.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGVI. Preparing the probe

◦Small piece of DNA used to find another piece of DNA

◦Must be labeled to be visualized◦Usually prepared by making a radioactive copy of a DNA fragment.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThe DNA fragment is labeled by

the Random Hexamer Labeling Process:◦1. The template DNA is denatured by boiling.

◦2. A mixture of hexamers (6 nucleotides) containing all possible sequences is added and allow to base pair.

SOUTHERN BLOTTINGSOUTHERN BLOTTING◦3. DNA polymerase is added with radioactive nucleotides.

◦4. The mixture is boiled to separate the strands and is ready for hybridization.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGThe Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

SOUTHERN BLOTTINGSOUTHERN BLOTTING

SOUTHERN BLOTTINGSOUTHERN BLOTTINGVII. Hybridization

◦The labeled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGVIII. Washing

◦Excess probe will have bound nonspecifically to the membrane despite the blocking reagents.

◦Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGIX. Detection

◦Radioactive probes enable autoradiographic detection.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGIf the probe is radioactive, the

particles it emits will expose X-ray film.

By pressing the filter and film, the film will become exposed wherever probe is bound to the filter.

After development, there will be dark spots on the film wherever the probe bound.

SOUTHERN BLOTTINGSOUTHERN BLOTTINGSummary of procedure

◦ 1. Extract and purify DNA from cells◦ 2. DNA is restricted with enzymes◦ 3. Sort by electrophoresis◦ 4. Denature DNA◦ 5. Transfer to nitrocellulose paper◦ 6. Block with excess DNA◦ 7. Wash off unbound probe◦ 8. Autoradiograph

Watch pointsWatch pointsUsing too little DNA-compromise

the sensitivity of the testUsing too much DNA- poor

restriction enzyme digestionUsing too high voltage setting for

electrophoresis- gel to melt or appearance of artifacts

Watch pointsWatch pointsImproper blocking-high

background and uninterpretable results.

Insufficient washing-high background and uninterpretable results.

Excess washing- dissociate the specific hybrids.

USESUSESIdentify mutations, deletions, and

gene rearrangementsUsed in prognosis of cancer and

in prenatal diagnosis of genetic diseases

LeukemiasDiagnosis of HIV-1 and infectious

disease

USESUSESEvery person has repeated

sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs)

To find a particular VNTR we use a radioactive version of the one in question.

This pattern is known as a DNA fingerprint.

USESUSESApplications of DNA

fingerprinting include:◦Paternity and Maternity Testing◦Criminal Identification and Forensics◦Personal Identification

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