Blotting techniques
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Transcript of Blotting techniques
M.Prasad NaiduMSc Medical Biochemistry,
Ph.D.Research Scholar
Definition
Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules requires the convergence of number of techniques which are collectively termed BLOT transfer .
Types of blotting techniques
1 ) Southern blotting ( to detect DNA )2 ) Northern blotting ( to detect RNA )3 ) Western blotting ( to detect protein )
Southern BlottingIn 1975 Edward Southern developed this
technique that is widely used to detect fragments of DNA .
This requires 1 ) Separation of DNA or DNA fragments by agarose gel electrophoresis .2 ) DNA fragments are blotted onto a strip of nitrocellulose or a nylon membrane.3 ) Identification by hybridization with a labeled ,complementary nucleic acid probe.
Definition Southern blot a method for transferring DNA
from an agarose gel to nitrocellulose filter , on which the DNA can be detected by suitable probe ( eg : complementary DNA or RNA ) .
Procedure The DNA sample is digested by restriction endonucleases , producing small fragments & that are amenable for analysis .
Fragments are seperated by agarose gel electrophoresis or PAGE .
The mobility of nucleic acids in agarose gels is influenced by agarose concentration , molecular size & molecular conformation of the nucleic acid .
Agarose concentration of 0.3 – 2 %are most effective for nucleic acid separation .
Like proteins nucleic acids migrate at rate that is inversely proportional to the logarithm of their molecular weight .
contdSeparated nucleic acids are visualized by
fluorescent dye ethidium bromide .The agarose gel is soaked in a solution of dye
& washed for remain excess dye .illumination of the rinsed slab with UV light
reveals red orange stains where nucleic acids are located .
contdEthidium bromide stains both single & double stranded nucleic acids , the fluorescence is much greater with double stranded molecules .
The electrophoresis can be performed with dye incorporated in the gel & buffer .
This has the advantage that the gel can be illuminated with UV light during electrophoresis to view the extent of separation.
contdThe mobility of DNA may be reduced by 10 -
15 % in the presence of ethidium bromide .
Ethidium bromide must be used with great care as it is a potent mutagen .
Gloves should be worn at all times while using the dye solutions or handling gels .
contdNewer fluorescent SYBR dyes produced by
molecular probes offer several advantages , less toxic & 5 times more sensitive than ethidium bromide.
Labeled DNA with radioisotope P32 at 5’ & 3’ ends .
P 32 is a strong β emitter .Bands of labeled DNA on electrophoresis gel
can located by autoradiography .
contdLabelling molecule before analysis with
coenzyme biotin , biotin forms a strong complex with enzyme linked streptavidin .
PAGE is useful for analysing small fragments of DNA upto 3,50,00 daltons ( 500 bp ) in molecular size .
Large molecules of DNA could be separated by pulsed field gel electrophoresis.
BlottingTransfer of DNA from gel to nitrocellulose
membrane done by 1 ) Weak acid treatment to depurinate &fragment the DNA , thus make it smaller & easier to elute from the gel .
followed by2) Denaturate with strong base& neutralisation ( hydrolyzes phosphodiester back bone at depurinated sites )
single strands bind to membranes more efficiently )
A buffer is used to facilitate the transfer .
contdOriginal methods of transfer relied on
capillary action .Vaccum or preassure systems can be used to
speed the transfer .Faster & more efficient transfer is afforded
by the use of an electroblotter .Electroblotting process is usually completes
in 1-4 hours .
Hybridization assaysAll hybridization assays are based on the ability of nucleic acids to form specific double stranded hybrids .
The process requires 1 ) A probe that can target nucleic acids &
allow for specific complemenatary base pairing .2) A method to detect any resulting double strands nucleic acids .
contd Conditions of high stringency in
hybridization assay are 1) Low salt concentration ,2) High formamide levels ,3) High temparature . As the stringency of the assay is lowered
increasing number of base mismatches are tolerated .
conditions of high stringency require exact base pairing .
The time required to hybridize the probe to a given fraction of the target remains proportional to the probe concentration .
The rate of hybridization reaction is influenced by temperature & ionic strength.
Above the Tm no stable hybrids are present .Divalent cations like Mg+2 have stronger
effect on hybridization .
contd
Unbound probes are removed by washing
Probe bound to the membrane is detected by autoradiogarphy , which reveals the DNA fragments to which the probe hybridized .
Applications Southern blots are used in gene discovery,
mapping , evolution & development studies , diagnostics & forensics .
Deletions / insertions .pointmutations / polymorphisms .Structural rearrangements .Allow for determination of molecular
weights of restriction fragments .Presence of particular bit of DNA in the
sample.
Northern blotting Northern blotting is a technique for
detection of specific RNA sequences .Developed by James alwine & George stark.
RNA molecules have defined length & much shorter than genomic DNA it is not necessary to cleave RNA before electrophoresis .
RNA is more susceptible to degradation than DNA .
RNA sample are separated based on size by gel electrophoresis .
contdRNA is blotted on to a nylon positively
charged membrane .The membrane is placed in a hybridization
buffer with a labeled probe ( usually DNA )Labeled probe is detected by
autoradiography Expression patterns of sequences of interest
in different samples can be compared .
ApplicationsA standard for direct study of the gene
expression at the level of mRNA .
Detection of mRNA transcript size .
Study of RNA splicing – can detect alternatively spliced transcripts .
Study RNA half life
Disadvantages Time consuming procedure .
RNA samples can be degraded by RNases .
Use of radioactive probes .
Detection with multiple probes is a problem .
Western blotting Western blotting is an immunoblotting technique which rely on the specificity of binding between the molecule of interest & a probe to allow detection of molecule of interest in a mixture of many other similar molecules .
In western blotting the molecule of interest is a protein & the probe is typically an antibody raised against that particular protein .
Contd SDS PAGE technique is a prerequisite for
western blotting .
Protein sample is subjected to electrophoresis on SDS polyacrylamide gel .
Electroblotting transfers the separated proteins from the gel to the surface of nitrocellulose membrane .
contdBlot is incubated with generic protein
( such as milk protein )which binds to any remaining sticky places on the nitrocellulose .
An antibody which is specific for the protein of interest ( the primary antibody Ab 1 ) is added to the nitrocellulose sheet & reacts with the antigen . Only the band containing protein of interest binds the antibody forming a layer of antibody molecules .
contd
Following several rinses for removal of nonspecifically bound Ab1 , the Ab1 – antigen complex on the nitrocellulose sheet is incubated with second antibody Ab2 , which specifically recognizes the Fc domain of the primary antibody & binds it . Ab 2 is radioactive labeled or is covalently linked to reporter enzyme which allows to visualize protein – Ab1 – Ab2 complex .
Applications The confirmatory HIV test employs a western
blot to detect anti HIV antibody in a human sample .
Proteins from known HIV infected cells are separated & blotted on a membrane then the serum to be tested is applied in the primary antibody incubation step.
Free antibody is washed away & a second anti human antibody linked to an enzyme signal can be added .
contdThe stained bands then indicate the proteins
to which the patient serum contains antibody .
Western blot is also used as definitive test for bovine spongiform encephalopathy . ( mad cow disease )
Some forms of Lyme disease testing employs western blotting .