Assignment

Recombinant DNA
Technology
Topic: Northern Blotting
Submitted to: Dr. Ameer Khan
Submitted by: Mujahid Hussain
Roll Number: 12
Class: M.Phil
1 Roll No. 12 (Mujahid Hussain) M.Phil Botany 12/2/2016
 Blotting

 Process in detecting any

macromolecule that we deal with
it
 Macromolecule may be DNA, RNA
or Protein

2 Roll No. 12 (Mujahid Hussain) M.Phil Botany 12/2/2016

 Blotting
 IF we are detecting DNA we called
it Southern Bloting
 IF we are detecting RNA we called
it Nothern Bloting
 IF we are detecting Protein we
called it Western Boting

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 Northern Blotting

 Technique developed in 1977 or 1979 by

J.Alwine, D. Kemp & G. Start

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 Northern Blotting

 Very Similar to Southern Bloting

 Technique based on Nucleic acid
Hybridization
Nucleic acid hybridization: A technique in which single-
stranded nucleic acids(DNA or RNA) are allowed to interact so
that complexes called hybrids are formed by molecules with
similar, complementary sequences.

5 Roll No. 12 (Mujahid Hussain) M.Phil Botany 12/2/2016

 Northern Blotting (Importance)

 To Study gene expression by detecting RNA

in a Sample During differentiation,
morphogenesis as well as abnormal or
diseased condition

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 Northern Blotting (Importance)

 Detects the presence a specific mRNA

in a total RNA extract
 Can determine whether a gene is
transcribed or not

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 Northern Blotting (Stages)
 Major stages involve in Northern Bloting

 To Prepare target
 Gel electrophoresis
 Blot
 Hybridization
 Detection

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 Northern Blotting (Process)

 To Prepare target
 Target is RNA

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 Northern Blotting (Process)

 Simply Extract the content of cell that contain

RNA and than Purify RNA Extract from other cell
components
 RNA Extraction and purification takes place by
Trizol

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 Northern Blotting (Process)

 After Extraction and Purification

 Treat RNA with different endonucleases

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 Northern Blotting (Process)

 Endonulceases are Restriction endonucleases

 Cut the RNA into different Fragments

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 Northern Blotting (Process)

 But the cutted Fragments of RNA are present in

Secondary Structure because whenever the find
complementary regions, they bind

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 Northern Blotting (Process)

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 Northern Blotting (Process)

 For Detection they must be Linear

 TO Solve this problem we use DENATURING
GEL ELECTROPHORESIS

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 Northern Blotting (Process)

 DENATURING GEL ELECTROPHORESIS

 Agarose gel with extra formaldehyde is used for
this purpose
DENATURING GEL
ELECTROPHORESIS
Separate the fragments on the basis
of size
And linear the RNA

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 Northern Blotting (Process)

 Extra Formaldehyde makes it (RNA Fragments)

linear

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 Northern Blotting (Process)

 Smaller Fragments move faster, migrate more,

travel more distance
 Larger Fragments move slower, migrate less,
travel less distance

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 Northern Blotting (Process)

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 Northern Blotting (Process)

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 Northern Blotting (Process)

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 Northern Blotting (Process)

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 Northern Blotting (Process)

 After the Separation of fragments on the basis of

size
 We will go for Blotting

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 Northern Blotting (Process)

 Blotting
 IMPRINTING gel material on to the paper

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 Northern Blotting (Process)

 In Blotting
 We Transfer the content of gel onto the paper

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 Northern Blotting (Process)

 In this Blotting
 We use Whitman's Filter paper No. 52
 Amino Benzoxy Methyl In Place of Nitrocellulose
membrane Filter Paper (Southern Blotting)

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 Northern Blotting (Process)

 Amino Benzoxy Methyl

 Better transfer medium for RNA
 More binding affinity towards RNA

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 Northern Blotting (Process)

 Transfer the content of gel onto the paper is takes

place by through capillary action

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 Northern Blotting (Process)

 Capillary action transfer the content of gel (RNA)

from gel to the paper (Amino benzoxy methyl)

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 Northern Blotting (Process)

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 Northern Blotting (Process)
Weight
Amino Benzoxy
Me.

31 Roll No. 12 (Mujahid Hussain) M.Phil Botany Amino benzoxy 12/2/2016

methyl
 Northern Blotting (Process)

 After transferring the content of gel (RNA) from

gel to the paper (Amino benzoxy methyl)
 We Exclude everything out and take membrane
out

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 Northern Blotting (Process)

 Put the membrane into a solution containing

probes

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 Northern Blotting (Process)

 Probe will Hybridize only on a specific Target

mRNA
 In case of Southern Bloting DNA-DNA duplex is
formed
 In case of Northern Bloting RNA-DNA duplex is
formed Probe
Short sequence of Nucleotide that is
complementary to its target

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 Northern Blotting (Process)

 Bath the membrane with another solution that

would not be containing a probe
 BY THIS WAY all probes will be removed except
target hybridized probe

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 Northern Blotting (Process)

 After probe Hybridization

 We will go for Detection

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 Northern Blotting (Process)

 IF probe is radioactively labeled, we go for Radio

autography
 IF probe is Fluorescent, we go for
chemiluminescence
 IF probe is Colori, we go for colorimetric analysis

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 Northern Blotting (Process)
 IF probe is radioactively labeled, we go for Radio
autography We put X-ray
Band is formed on X-ray

 IF probe is Fluorescent, we go for

Probe will Emit Light
chemiluminescence

Probe Will Emit color

 IF probe is Colori, we go for colorimetric analysis

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 Northern Blotting (disadvantages)

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 Comparison Between
Northern Blotting & Southern Blotting
Southern Blotting Northern Blotting
 DNA molecules  RNA molecules detected

detected  Agarose denaturing gel

 Agarose gel electrophoresis
 DNA/DNA hybridization
 Detecting systems are
Colori, Radioactive,
Chemical
 Probe is ssDNA
 Blotting method is capillary
action
Roll No. 12 (Mujahid Hussain) M.Phil Botany
40
 Nitrocellulose membrane is
electrophoresis with extra
formaldehyde
 DNA/RNA hybridization
 Detecting systems are
Colori, Radioactive,
Chemical
 Probe is ssDNA
 Blotting method is capillary
action
 Amino benzoxy methyl
membrane is used 12/2/2016
 RNA EXTRACTION
BY TRIZOL

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 RNA EXTRACTION BY TRIZOL
(Process)

 Add 1ml trizol to the sample and homogenize

 Add 2ul chloroform to homogenate
 Vortex vigorously

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 RNA EXTRACTION BY TRIZOL

 Incubate on ice for 15 minutes

 Centrifuge to get phase separation
 12,000g for 15minutes at 4 degree centigrade

g is the relative
centrifugal force

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 RNA EXTRACTION BY TRIZOL
 Transfer the aqueous phase to a fresh tube

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 RNA EXTRACTION BY TRIZOL

 Precipitate the RNA by mixing with 0.5ml

isopropanol
 Incubate on ice for 10 minutes
 Centrifuge for 10minutes at 12,000g at 4 degree
centigrade

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 RNA EXTRACTION BY TRIZOL
 Remove the supernatant (Surface Liquid)
 Wash pellet with 1ml 70% ethanol by flicking
 Centrifuge at 7500g for 10minutes at 4 degree
centigrade

Supernat
ant

Pellet

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 RNA EXTRACTION BY TRIZOL

 Remove supernatant
 Air dry the Pellet (RNA Pellet)
 Dissolve RNA Pellet in appropriate volume of
Rnase-free water
Now further we treat this RNA with restriction
endonucleases
And cutted into Fragments

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 Summary (NORTHERN BLOTTING)

 Prepared target ( Fragmented RNA)

 Gel electrophoresis
 Blotting
 Hybridization
 Detection

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  Thank You 

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