genetics and molecular biology

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2. 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 .

3. Types of blotting techniques
1 ) Southern blotting ( to detect DNA )
2 ) Northern blotting ( to detect RNA )
3 ) Western blotting ( to detect protein )

4. Southern Blotting
In 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.

5. 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 ) .

6. 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 .

7. 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 .

8. contd
Separated 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 .

9. contdEthidium 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.

10. contdThe 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 .

11. contdNewer 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 .

12. contdLabelling 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.

13. Blotting
Transfer 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 by
2) 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 .

14. contdOriginal 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 .

15. Hybridization assaysAll 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 .

16. 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 .

17. 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 .

18. 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 .

19. 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.

20. 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 .

21. contdRNA 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 .

22. Applications
A 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

23. Disadvantages
Time consuming procedure .
RNA samples can be degraded by RNases .
Use of radioactive probes .
Detection with multiple probes is a problem .

24. 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 .

25. 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 .

26. contd
Blot 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 .

27. 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 .

28. 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 .

29. contd
The 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 .