- Electrophoresis separates nucleic acids and proteins based on size. Nucleic acids move towards the positive electrode through agarose or acrylamide gels. Agarose gels separate larger fragments, while acrylamide separates smaller ones.
- Restriction enzymes cut DNA at specific palindrome recognition sequences, producing fragments of predictable average sizes. Southern blots detect specific DNA sequences within restriction fragments separated by gel electrophoresis.
- Electrophoresis followed by blotting and probing allows detection and analysis of specific genes, RNAs, and proteins.
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive labelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminiscent reaction which is registered by photographic film.
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.[1]
Western blot
A Western blot is used for the detection of specific proteins in complex samples. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix (usually PVDF or nitrocellulose) and subsequent detection with antibodies.
Eastern blot
The Eastern blot is used for the detection of specific posttranslational modifications of proteins. Proteins are separated by gel electrophoresis before being transferred to a blotting matrix whereupon posttranslational modifications are detected by specific substrates (cholera toxin, concanavalin, phosphomolybdate, etc.) or antibodies.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
Genetic Organisation:
All cellular activities are encoded within a cell’s DNA.
The sequence of bases within a DNA molecule represents the genetic information of the cell.
Segments of DNA molecules are called genes, and individual genes contain the instructional code necessary for synthesizing various proteins, enzymes, or stable RNA molecules.
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive labelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminiscent reaction which is registered by photographic film.
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.[1]
Western blot
A Western blot is used for the detection of specific proteins in complex samples. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix (usually PVDF or nitrocellulose) and subsequent detection with antibodies.
Eastern blot
The Eastern blot is used for the detection of specific posttranslational modifications of proteins. Proteins are separated by gel electrophoresis before being transferred to a blotting matrix whereupon posttranslational modifications are detected by specific substrates (cholera toxin, concanavalin, phosphomolybdate, etc.) or antibodies.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
Genetic Organisation:
All cellular activities are encoded within a cell’s DNA.
The sequence of bases within a DNA molecule represents the genetic information of the cell.
Segments of DNA molecules are called genes, and individual genes contain the instructional code necessary for synthesizing various proteins, enzymes, or stable RNA molecules.
Recombinant dna technology and DNA sequencinganiqaatta1
title: recombinant DNA technology and DNA sequencing
this lect will cover the pcr, isolation of DNA, detection of DNA and DNA manipulation joining DNA together. this is very important and it is required in research of every field especially medical related field.
The topic explains briefly on central dogma of molecular biology, DNA packaging in chromosome, to understanding the nature of genetics Code and to compare the mitochondria & chloroplast DNA with nuclear DNA
Molecular biology is a branch of science concerning biological activity at the molecular level.
The field of molecular biology overlaps with biology and chemistry and in particular, genetics and biochemistry.
A key area of molecular biology concerns understanding how various cellular systems interact in terms of the way DNA, RNA and protein synthesis function.
Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
Vasundhara Hospital & Fertility Research Centre is advanced IVF speciality centre in Jaipur, Rajasthan at very low cost with experienced team. Book your appointment now!
Get more info :- https://www.vasundharafertility.com/jaipur
Recombinant dna technology and DNA sequencinganiqaatta1
title: recombinant DNA technology and DNA sequencing
this lect will cover the pcr, isolation of DNA, detection of DNA and DNA manipulation joining DNA together. this is very important and it is required in research of every field especially medical related field.
The topic explains briefly on central dogma of molecular biology, DNA packaging in chromosome, to understanding the nature of genetics Code and to compare the mitochondria & chloroplast DNA with nuclear DNA
Molecular biology is a branch of science concerning biological activity at the molecular level.
The field of molecular biology overlaps with biology and chemistry and in particular, genetics and biochemistry.
A key area of molecular biology concerns understanding how various cellular systems interact in terms of the way DNA, RNA and protein synthesis function.
Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
Vasundhara Hospital & Fertility Research Centre is advanced IVF speciality centre in Jaipur, Rajasthan at very low cost with experienced team. Book your appointment now!
Get more info :- https://www.vasundharafertility.com/jaipur
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
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All of this illustrated with link prediction over knowledge graphs, but the argument is general.
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This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
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This talk is aimed at encouraging a more independent approach to using PHP frameworks, moving towards a more flexible and future-proof approach to PHP development.
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The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
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Cheryl Hung, ochery.com
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Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
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Here is something new! In our next Connector Corner webinar, we will demonstrate how you can use a single workflow to:
Create a campaign using Mailchimp with merge tags/fields
Send an interactive Slack channel message (using buttons)
Have the message received by managers and peers along with a test email for review
But there’s more:
In a second workflow supporting the same use case, you’ll see:
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And...
Speakers:
Akshay Agnihotri, Product Manager
Charlie Greenberg, Host
2. Principles recap
• Nucleic acids (DNA, RNA) move from the negative
to the positive pole during gel electrophoresis
• The resistance to movement is due to the sieving
effect of the gel matrix
• The rate of migration is related to the size of the
nucleic acid
3. Agarose versus acrylamide
• Acrylamide is a crosslinked synthetic polymer useful in
separating DNA molecules of a few hundred bases in length
– The crosslinking reaction is carried out within the molding
apparatus
– The final gel is transparent and usually used in a vertical apparatus
• Agarose is a polysaccharide similar to starch useful in
separating DNA and RNA molecules of a few hundred to
tens of thousands on bases long
– A gel is created by boiling agarose into solution and allowing it to
cool within a molding apparatus
– The final gel is translucent and usually used in a horizontal
apparatus
4. Restriction enzymes I
• Origin and native utility
– Some restriction enzymes are used by bacteria to
protect themselves from invading DNA
– A “restriction-modification” system exists that marks
bacterial DNA by methylating it on certain sequences
• A restriction enzyme specific for those sequences cannot
digest DNA if it is methylated
– Invading viral DNA is unmarked and subject to
cleavage at those sequences
5. Enzymes
• Restriction enzymes
– Generally bacterial in origin
• But may come from lower
eukaryotes, viruses or transposable
elements
– Cleave DNA at short
palindrome sites
• The palindromes may be 2 or more
nucleotides long
• Unusual forms may recognize
sequences up to 30 bases long
– These are used in site specific
recombination events
• Cleavage usually results in a 5’
phosphate and a 3’ hydroxyl
• The resulting ends may be single
stranded over a short region, or
double stranded (blunt ends)
• The cleavage sites may be
covalently linked back together by
DNA ligase
6. Restriction enzymes III
• A restriction enzyme with a 6 base recognition site
would produce on average a fragment size of 4096
bases in length when used to cut single copy DNA
– The average fragment size can be determined for any
recognition site by the formula 4n
where n is the number
of bases in the recognition site
Size of recognition
sequence
Average size of
fragment
4 256
5 1024
6 4096
8 65536
7. Agarose gel
electrophoresis
• Gels are loaded with samples
of restricted DNA and
electrophoresed for minutes to
hours
• The voltage is turned off and
the gel is stained with the
intercalator ethidium bromide
– DNA may be visualized in the
gel by illuminating it with
ultraviolet light
– Linear relationship between
mobility and the logarithm of
the DNA size
8. Size determination
• Size markers are commonly
used in electrophoresis
experiments
– These are duplex DNA
fragments of a known size
– The migration distance of a
fragment may be compared to
the migration of the size markers
directly on the gel
– Alternatively the migration of a
fragment may be determined by
interpolation from a plot of the
distance migrated by the size
markers
• The distance migrated is
related to the inverse log of the
molecular weight of the DNA
fragment
Migration versus size
100
1000
10000
100000
0 2 4 6 8
Distance migrated in cm
Nucleotidebasepairs
9. Digesting human
chromosomal DNA
• produces a range of fragment
sizes due to cleavage of single
copy DNA
– Three billion base pairs cleaved by
a restriction enzyme recognizing 6
bases results in 750,000 fragments
• They appear as a background
smear of DNA cleaved into all
possible sizes by the restriction
enzyme
• The fragments are of random size
with an average length of >4096
bases
– Repetitive sequence can increase
the average size, because usually
a given restriction site will not
be found within it
– If a restriction site is in a repetitive
sequence, digestion will produce a
large number of fragments of
identical length
• This will appear as a band in the
background smear of fragments
10. Southern
Blot
• Once an agarose gel of digested chromosomal DNA
has been stained, individual DNA sequences may be
detected on the gel by hybridization
– The DNA within the gel is denatured by exposing it to a
solution of sodium hydroxide
– The DNA is then neutralized and transferred out of the gel
onto a membrane that binds DNA
• This is a procedure known as blotting
• It exposes the DNA to the surface so that it may hybridize to
complementary sequences
– The membrane bound DNA is then hybridized to a short
specific sequence known as a probe
11. Probe
• A probe is an oligo or poly nucleotide that
represents known DNA sequence
– It is usually a sequence complementary to a
gene
• But it only need be complementary to DNA
sequence within the chromosomal DNA itself
– It is either single stranded to begin with or is
denatured to make it single stranded
– It is labeled with radioactive phosphorous or a
fluorescent adduct
12. Hybridization
• The membrane is
“prehybridized” with non-
specific DNA to block non-
specific binding sites on the
membrane prior to
hybridization
• It is then hybridized to the
probe
– The temperature and salt
conditions are controlled to insure
optimal hybridization between the
probe and the target sequences
– The single stranded probe finds
its complement in the membrane
bound DNA and forms a double
helix
• Unbound probe is then washed
off
13. X-ray film
exposure
• The membrane is exposed to
X-ray film
– Radioactive probes expose the
film whereever they hybridized
on the blot due to emission of
electrons (beta particles)
– Fluorescent probes catalyze a
light yielding reaction using
energy supplied in a reaction
cocktail
• Development of the X-ray
yields an autoradiogram
– Bands on the film locate the
chromosomal restriction
fragments complementary to the
probe DNA
14. Overall
• Southern Blots
– DNA is digested with restriction enzymes and
electrophoresed through an agarose gel
– The contents of the gel are denatured in situ
and then transferred onto a membrane that
binds DNA
– The DNA is hybridized to radioactive DNA
complementary to the gene of interest
– Unhybridized DNA is washed away and the
membrane exposed to X-ray film
• The technique can be adapted to identify
any source of DNA containing a gene of
interest or to RNA detection (northern
blots)
15. Restriction fragment length
polymorphism (RFLP)
• Chromosomal DNA from two people differs due
to a random variations in sequence
– There are millions of single nucleotide polymorphisms
between two unrelated individuals
• If one of these sequence variations results in the
creation or destruction of a restriction site, then
the fragment sizes of DNA will differ between
these two people
• This can be detected by southern blots
16. Example I RFLP point mutation
• A gene sequence contains two Eco R1 sites
separated by 4000 nucleotides
• A patient has suffered a mutation in one of his two
homologous chromosomes such that a single
nucleotide change has created a new EcoR1 site
within the gene sequence
– Now two fragments are created of 1500 and 2500 bases
in length
– The probe is complementary to sequence located within
the 2500 base long fragment
– Detection by southern blot of these two fragments will
result in a 4000 base long fragment in normal DNA
from one homologous chromosome and two fragments
from the other homolog
17. DNA from normal individual
digested with Eco R1 and
electrophoresed through agarose
DNA from mutant individual digested
with Eco R1 and electrophoresed
through agarose
20. Other blots Northern
• Northern blots are mainly used to measure of the
amount of a specific RNA in the cell
• The blot is prepared and hydridized like a
southern blot
• There are a few minor technical alterations
– The RNA is usually folded into a secondary structure
so it must be denatured before and during
electrophoresis
• Bands on the autoradiogram are a measure of the
presence of RNA from a particular gene
21. Protein electrophoresis
• Proteins, unlike DNA, do not have a constant size
to charge ratio
– In an electric field, some will move to the positive and
some to the negative pole, and some will not move
because they are neutral
– Native proteins may be put into gel systems and
electrophoresed
• However native proteins are sometimes difficult to prepare
without degradation
– An alternative to native protein gels forces all proteins
to acquire the same size to charge ratio
22. SDS-polyacrylamide gel
electrophoresis
• A sample of protein, often freshly isolated and
unpurified, is boiled in the detergent sodium
dodecyl sulfate and beta-mercaptoethanol
– The mercaptoethanol reduces disulfide bonds
– The detergent disrupts secondary and tertiary structure
– On the molecular level, proteins are stretched out and
coated with the detergent (which has a negative charge)
by this treatment
– They will then migrate through a gel towards the positive
pole at a rate proportional to their linear size
• Molecular weights with respect to size markers may then be
determined
23. Other blots II
Western
• Western blots measure the amount of
a specific protein in a cell
• This is a blot of an SDS-
polyacrylamide gel onto which
cellular protein is applied
– The protein is denatured so it will enter
the gel as a stretched out random coil
– The proteins separate by size
– They are blotted onto a membrane
• Western blots are probed using
antibodies
– The antibodies are labeled. Those that
remain bound to their specific targets
identify specific proteins in the cell
• The intensity of the signal reflects
the level of expression
• It can also reveal mutations by
anomalies of migration
– A deletion might cause a shorter protein
for example