Northern blotting is a technique used to detect specific RNA sequences. It involves separating RNA samples by electrophoresis in a gel, transferring the RNA to a membrane, and using a labeled probe to detect the RNA of interest. Western blotting is similar but detects specific proteins using antibodies as probes. It involves separating proteins by electrophoresis, transferring them to a membrane, and using primary and secondary antibodies to detect the protein of interest. Both techniques are used to study gene and protein expression.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Dr. S. MANIKANDAN, M.Sc., Ph.D
Lecturer in Botany
Thiruvalluvar University Model Constituent College,
Tittagudi 606 106, Tamil Nadu, India.
Email id: drgsmanikandan@gmail.com
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Dr. S. MANIKANDAN, M.Sc., Ph.D
Lecturer in Botany
Thiruvalluvar University Model Constituent College,
Tittagudi 606 106, Tamil Nadu, India.
Email id: drgsmanikandan@gmail.com
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
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Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
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neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
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and severe subclassifications (American Psychiatric Association, 2013).
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combined into a single substance use disorder (SUD) on a continuum
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comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
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TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
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Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
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RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
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MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdf
Lecture 4 northern and western blotting 2
1. Dr. Vishnu Kumar
Professor, Department of
Biochemistry, SRMSIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
Northern & Western
Blotting Techniques
3. Northern and western blotting
• This is conceptually similar to Southern
blotting. Here RNA is subjected to
electrophoresis before blot transfer.
• In Western blot proteins are separated
by electrophoresis and blot transfer is
performed. Here the probe will be
antibodies.
4. Probes.
• To search a desired DNA sequence of
interest from a vast array of DNA
fragments, a reagent is needed which
would react only with the correct
fragment and ignore the rest.
• This type of reagents are called
probes. It is a single strand of DNA or
RNA which can base pair with a
specific DNA, RNA or protein fragment.
5. Retrovirus
• Definition they are RNA virus and are
distinguished from other RNA virus by
their ability to replicate through a DNA
intermediate using an enzyme called
reverse transcriptase (R.T.)
• R.T. allows viral RNA to be transcribed
into DNA which is then incorporated
into the host cell genome.
6. Transmission of retro virus
• HIV and oncogenic virus enter the host
cell.
• Viral RNAR.T. cDNA, cDNA- viral
RNA hybrid RNAse H cDNA, RNA is
released.
• cDNA acts as a template on which new
DNA strand is synthesized. So a double
stranded DNA is formed which contains
the sequence of viral RNA.
7. Genome of retrovirus.
• They are RNA virus containing reverse
transcriptase. Retro virus genome
contains four genes gag, pol. Env, and
src.
• Gag codes for viral antigen.
• Pol codes for reverse transcriptase.
• Env codes for glycoprotein of viral
envelope.
• Src codes for or a protein kinase.
8. Reverse Transcriptase
• An RNA dependent DNA polymerase.
• It is the special feature of a retro virus. It is
complexed to an RNA in the viral core.
• It catalyzes the transcription of RNA genome
into a double stranded DNA that migrates
from cytoplasm to the nucleus and integrates
into the host cell DNA.
• Integration is helped by the enzyme,
integrase.
9.
10.
11. Northern Blotting
Northern blotting is a technique for detection of
specific RNA sequences.
Northern blotting was developed by James Alwine
and George Stark at Stanford University (1979)
and was named such by analogy to Southern
blotting.
12. Steps involved in Northern blotting
s
• 1. RNA is isolated from several biological
samples (e.g. variou tissues, various
developmental stages of same tissue etc.)
• * RNA is more susceptible to degradation
than DNA.
13. Cont……
2. Sample’s are loaded on
gel and the RNA samples are
separated according to their
size on an agarose gel .
The resulting gel
following
after the electrophoresis run.
14. Cont……
3. The gel is then blotted on
a nylon membrane or a
nitrocellulose filter paper
by creating the sandwich
arrangement.
15. 4. The membrane is placed in a dish
containing hybridization buffer
with a labeled probe.
Thus, it will hybridize to the RNA
on the blot that corresponds to
the sequence of interest.
5. The membrane is washed to
remove unbound probe.
16. 6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is
used).
In both cases this results
in the formation of a dark band on
an X-ray film.
Now the expression patterns of
the sequence of interest in the
different samples can be
compared.
17. APPLICATIONS
A standard for the study of gene expression at
the level of mRNA (messenger RNA transcripts)
Detection of mRNA transcript size
Study RNA degradation
Study RNA splicing
Study RNA half-life
Often used to confirm and check transgenic /
knockout mice (animals)
18. Disadvantage of
Nourthern plotting
1.The standard northern blot method is
relatively less sensitive than nuclease
protection assays and RT-PCR
2.Detection with multiple probes is a problem
3.If RNA samples are even slightly degraded
by RNAses, the quality of the data and
quantitation of expression is quite
negatively affected.
19. Western blotting
Western blotting (1981) is an Immunoblotting
technique which rely on the specificity of binding
between a protein of interest and a probe
(antibody raised against that particular protein) to
allow detection of the protein of interest in a
mixture of many other similar molecules.
The SDS PAGE technique is a prerequisite for
Western blotting .
20. Steps in western blotting
1. A protein sample is subjected
to electrophoresis on an
SDS-polyacrylamide gel.
2. Electroblotting transfers the
separated proteins from the
gel to the surface of a
nitrocellulose membrane.
21. Cont…
3. The blot is incubated with a generic protein
(such as milk proteins or BSA) which binds to
any remaining sticky places on the
nitrocellulose.
4. An antibody that is specific for the protein of
interest (the primary antibody - Ab1) is added
to the nitrocellulose sheet and reacts with the
antigen. Only the band containing the
protein of interest binds the antibody,
forming a layer of antibody molecules .
22. Cont…
5. After washing for removal of non-
specifically bound Ab1, second
antibody (Ab2)is added, which
specifically recognizes the Fc domain
of the primary antibody and binds it.
Ab2 is radioactively labeled, or is
covalently linked to a reporter
enzyme, which allows to visualize the
protein-Ab1-Ab2 complex.
23.
24. Applications
1.The confirmatory HIV test
2.Western blot is also used as the
definitive test for Bovine spongiform
encephalopathy (BSE)
3.Some forms of Lyme disease testing
employ Western blotting .
25. LONG ANSWER QUESTIONS
1. Describe the Northern and Western Blotting
techniques and their applications.