Detection, analysis & characterization

of nucleic acid

Dr.Salma Mahmoud
 Measurement of Nucleic Acid Quality and
Quantity
 Laboratory analysis of nucleic acids produces variable results,
depending on the quality and quantity of input material.
 Resolution and detection of nucleic acids are done in several ways:
1. Electrophoresis
2. Spectrophotometry
3. Flourometry
 Gel and capillary electrophoresis are the most practical and frequently
used methods.
 DNA can also be spotted and detected using specific hybridization
probes
 The appearance of DNA on agarose gels depends on the type of
DNA isolated.
 A good preparation of plasmid DNA will yield a bright,
moderate-mobility single band of supercoiled plasmid DNA with
minor or no other bands that represent nicked or broken plasmid
 High-molecular-weight genomic DNA should collect as a bright
band with low mobility (near the top of the gel).
 A high-quality preparation of RNA will yield two distinct bands
of rRNA.
 The integrity of these bands is an indication of the integrity of the
other RNA species present in the same sample.
 If these bands are degraded (smeared) or absent, the quality of the
RNA in the sample is deemed unacceptable for use in molecular
assays.
 When fluorescent dyes are used, DNA and, less accurately, RNA
can be quantitated by comparison of the fluorescence intensity of
the sample aliquot run on the gel with that of a known amount of
control DNA or RNA loaded on the same gel.
 Densitometry of the band intensities gives the most accurate
measurement of quantity. For some procedures, estimation of
DNA or RNA quantity can be made by visual inspection.
Bands on a gel
 Electrophoresis

 DNA and RNA can be analyzed for quality by resolving an

aliquot of the isolated sample on an agarose gel
 Fluorescent dyes such as ethidium bromide or SybrGreen I
bind specifically to DNA and are used to visualize the sample
preparation.
 Ethidium bromide or SybrGreen II can be used to detect RNA.
Less frequently, silver stain has been used to detect small
amounts of DNA by visual inspection.
 Electrophoresis is the movement of molecules by an electric
current.
 This can occur in solution, but it is practically done in a matrix to
limit migration and contain the migrating material.
 Electrophoresis is routinely applied to the analysis of proteins and
nucleic acids.
 Each phosphate group on a DNA polymer is ionized, making
DNA a negatively charged molecule.
 Under an electric current, DNA will migrate toward the positive
pole (anode).
 When DNA is applied to a macromolecular cage such as agarose
or polyacrylamide, its migration under the pull of the current is
impeded, depending on the size of the DNA and the spaces in the
gel.
 DNA fragments will therefore migrate at speeds inversely related
to their size.
 Electrophoresis can be performed in tubes, slab gels, or
capillaries.
 Slab gel electrophoresis can have either a horizontal or vertical
format
 Gel Systems

 Gel matrices provide resistance to the movement of molecules

under the force of the electric current.
 They prevent diffusion and reduce convection currents so that the
separated molecules form a “band.”
 These matrices must be unaffected by electrophoresis, simple to
prepare and amenable to modification.
 Agarose and polyacrylamide are polymers that meet these criteria.
 Agarose Gels

 Agarose is a polysaccharide polymer extracted from seaweed.

 It is a component of agar used in bacterial culture dishes.
 Hydrated agarose gels in various concentrations, buffers, and sizes can
be purchased ready for use.
 Alternatively, agarose can be purchased and stored in the laboratory in
powdered form.
 For use, powdered agarose is suspended in buffer, heated, and poured
into a mold.
 The concentration of the agarose dictates the size of the spaces in the
gel and will, therefore, be determined by the size of DNA to be
resolved
 Small pieces of DNA (50–500 bp) are resolved on more
concentrated agarose gels, e.g., 2%–3%.
 Larger fragments of DNA (2000–50,000) are best resolved in
lower agarose concentrations,
 High-concentration agarose will impede migration, whereas very
low concentrations produce a weak gel with limited integrity.
 Typical agarose gel

Load samples in wells

xylene bromophenol
cyanol blue

-
+
 (the DNA fragments
are not visible
without some sort of
time of electrophoresis staining)
(progress monitored by marker dyes)
ethidium bromide-stained agarose gel

M samples

The marker lane (M) gives

size standards for
comparison with the sample
lanes
 Pulsed Field Gel Electrophoresis

 Very large, i.e., 50,000–250,000 bp, pieces of DNA cannot be

resolved efficiently by simple agarose electrophoresis.
 Even in the lowest concentrations of agarose, megabase
fragments are too severely impeded for correct resolution
(referred to as limiting mobility).
 For genomic-sized DNA molecules, pulses of current applied to
the gel in alternating dimensions enhance migration.
 Polyacrylamide Gels

 Very small DNA fragments and single-stranded DNA are best

resolved on polyacrylamide gels in polyacrylamide gel
electrophoresis (PAGE).
 Acrylamide, in combination with the cross-linker methylene
bisacrylamide , polymerizes into a gel that has consistent
resolution characteristics
 Polyacrylamide was originally used mostly for protein
separation, but it is now routinely applied to nucleic acid
analysis.
 Polyacrylamide gels are used for sequencing nucleic acids,
mutation analyses and other applications requiring the resolution
of nucleic acids down to the single-base level.
 Acrylamide is supplied to the laboratory in several forms like
powder or solution.
Polyacrylamide gel set up (protein gels)
Stacking gel: at low
pH, glycine is
protonated (no neg.
charge), Cl- ions at
the leading edge,
glycine trailing,
steep voltage
gradient in
between, that’s
where the proteins
get “focused” into a
thin band

Separating gel: at
higher pH, glycine
deprotonates, runs
with the Cl- at the
leading edge, and
the proteins
separate based on
size
 2/Spectrophotometry

 Nucleic acids absorb light at 260 nm through the adenine

residues.
 Using the Beer-Lambert Law, concentration can be determined
from the absorptivity constants (50 for DNA, 40 for RNA).
 The absorbance at this wavelength is thus directly proportional to
the concentration of the nucleic acid in the sample.
 Absorbance unit at 260 nm is equivalent to 50 mg/L (or 50 g/mL)
of DNA and 40 g/mL of RNA.
 To determine concentration, multiply the spectrophotometer
reading in absorbance units by the appropriate conversion factor.
 3/Fluorometry

 Fluorometry or fluorescent spectroscopy measures fluorescence

related to DNA concentration in association with DNA-specific
fluorescent dyes.
 Early methods used 3,5-diaminobenzoic acid 2HCl (DABA).
 This dye combines with alpha methylene aldehydes
(deoxyribose) to yield a fluorescent product. It is still used for the
detection of nuclei and as a control for hybridization and spot
integrity in microarray analyses.
 More modern procedures use the DNA-specific dye Hoechst
33258.
 This dye combines with adenine-thymine base pairs in the minor
groove of the DNA double helix and is thus specific for intact
double-stranded DNA. This binding specificity for A-T residues.
 Fluorometric determination of DNA concentration using Hoechst
dye is very sensitive
 PicoGreen and OliGreen are other DNA-specific dyes that can
be used for fluorometric Quantitation
 RNA may be measured in solution using SybrGreen II RNA gel
stain
 Fluorometry measurements require calibration of the instrument
with a known amount of standard before every run.
Analysis and Characterization of Nucleic Acids
and Proteins

 Nucleic acid analysis generally involves isolation and

characterization of the DNA or RNA sample of interest.

 Sample purification and quality assessment are important steps in

experimental workflows since the quality of the recovered nucleic
acid can affect the performance in downstream reactions.
 Restriction Enzyme Mapping

 Clinical and forensic analyses require characterization of specific

genes or genomic regions at the molecular level.
 restriction endonucleases provide a convenient tool for
molecular characterization of DNA.
 Restriction enzymes commonly used in the laboratory have four
to six base pair recognition sites, or binding/ cutting sites, on the
DNA.
 Any four to six base pair nucleotide sequence occurs at random
in a sufficiently long stretch of DNA.
 Therefore, restriction sites will occur naturally in DNA.
 Restriction site mapping, i.e., determining where in the DNA
sequence a particular restriction enzyme recognition site is
located, was initially developed using small circular bacterial
plasmids.
 The resultant maps were used to identify and characterize
naturally occurring plasmids and to engineer the construction of
recombinant plasmids.
 To make a restriction map, DNA is exposed to several restriction
enzymes separately and then in particular combinations.
 Take, for example, a linear fragment of DNA cut with the enzyme
PstI. After incubation with the enzyme, the resulting fragments
are separated by gel electrophoresis.
 The gel image reveals four fragments, labeled A, B, C, and D,
produced by PstI .
 From the number of fragments one can deduce the number of PstI
sites: three.
 The sizes of the fragments, as determined by comparison with
known molecular-weight standards, indicate the distance between
these sites or from the site to the end of the fragment.
 The pattern of fragments produced by restriction enzyme
digestion can be used to identify that DNA and to monitor certain
changes in the size, structure, or sequence of the DNA.
 Because of inherited or somatic differences in the nucleotide
sequences in human DNA, the number or location of restriction
sites for a given restriction enzyme are not all the same in all
individuals.
 The location and order of restriction enzyme sites on a DNA
fragment is a molecular characteristic of that DNA.
 The resulting differences in the size or number of restriction
fragments are called restriction fragment length
polymorphisms (RFLPs).
 RFLPs were the basis of the first molecular-based human
identification and mapping methods.
 RFLPs can also be used for the clinical analysis of structural
changes in chromosomes associated with disease (translocations,
deletions, insertions, etc.).
Nucleic Acid Hybridization

 These are procedures performed in the clinical molecular

laboratory for specific targets in genomic DNA.
 This requires visualization or detection of a specific gene or
region of DNA in the background of all other genes.
 There are several ways to find a particular region of DNA from
within an isolated DNA sample.
 The initial method for molecular analysis of specific DNA sites
within a complex background was the Southern blot.
 Modifications of the Southern blot are applied to analysis of RNA
and protein in order to study gene expression and regulation
 Nucleic Acid Hybridization

⚫These are methods by which nucleic acids may be identified:

⚫Southern hybridization
⚫Northern hybridization
⚫Western hybridization
⚫Colony or plaque hybridization
 Southern hybridization

⚫Southern hybridization was first developed by Edward M. Southern in the

mid-1970s

⚫Is a procedure based on the ability of a nucleic acid to base- pair with its

complementary sequence.

⚫It is also known as the Southern blotting technique.

 Principle
⚫Southern hybridization uses a nucleic acid with a known sequence, either a
DNA or RNA, as a probe.
 Probes:
 The probe for Southern and Northern blots is a single stranded
fragment of nucleic acid.
 The purpose of the probe is to identify one or more sequences of
interest within a large amount of nucleic acid.
 The probe therefore should hybridize specifically with the target
DNA or RNA that is to be analyzed.
 The probe can be RNA, denatured DNA, or other modified nucleic
acids.
  Label probe for later detection
 Radioactivity: The probe is labeled either radioactively so it can
be detected later by autoradiography
⚫Non-radioactive label: labeled with a small molecule (such as biotin)
so that it can be detected with an antibody.

⚫When using antibody , the antibody is usually covalently attached to an

enzyme (usually alkaline phosphatase or horseradish peroxidase),
which can cleave a compound to produce either a colored or a light-
emitting product (chemiluminescence).
⚫The target for the hybridization is a DNA sequence.
 Steps

⚫The first step in a genomic Southern hybridization would be to digest human genomic

DNA with a restriction enzyme

⚫This digested DNA is then applied to an agarose gel, which is a gelatin-like substance

to do electrophoresis

⚫In a genomic DNA digest, there are usually so many fragments that the result is simply

a smear of fragments, from very small to very large.

⚫After electrophoresis, we denature the double-stranded DNA by incubating

the agarose gel in sodium hydroxide (NaOH).

⚫ The resulting single-stranded DNA fragments must then be transferred

from the gel to another medium for hybridization(nitrocellulose filter
paper).

⚫The denatured DNA fragments will move toward the anode (positive

electrode), and when it comes in contact with the nitrocellulose, it binds.

⚫In the other approach, the NaOH-treated agarose gel is placed directly

against a piece of nitrocellulose filter, and the resulting sandwich is placed

upside- down on a wet sponge.

⚫Dry filter paper placed against the nitrocellulose side wicks a salt solution

from the sponge, through the gel, and past the nitrocellulose filter, carrying
the DNA segments from the agarose to the nitrocellulose

⚫The nucleic acid hybridization can now take place on the filter.

⚫The labeled cDNA or mRNA probe is denatured by heating and is added to

the nitrocellulose in a salt solution

Immobilization of nucleic acids

nitrocellulose
or nylon
membrane
boundary:
DNA binds to it

Agarose or
A typical capillary blotting apparatus. Polyacrylamide
Electroblotting is also commonly used gel
Southern Blot--one example

(or PCR fragment)

(RFLPs)
 Radioactive probes: 32P labeling
• Use T4 polynucleotide kinase
32Pis a high energy beta particle emitter, and provides good
sensitivity for detection of hybridization between the probe DNA
and the target (blot) DNA
• Detect radiolabel with
--autoradiography (X ray film)
--phosphorimager (phosphor coated plates store the energy of
the radioactive particle, laser excitation releases photons of
light that are collected and represented as a picture, greater
dynamic range than film, and faster too
Non-radioactive labels

…e.g. horseradish peroxidase

oxidation
 Non-radioactive labels
…or digoxygenin/antibody-conjugated HRP

oxidation

can also use

biotinylated
DNA probe
 Northern Hybridization

⚫It differs from the Southern, colony, and plaque hybridizations in that

RNA, not DNA, is the target.

⚫Use this method to measure levels of gene transcription in vivo

(detecting changes in the levels of RNA transcript under differing

conditions)
⚫In Northern hybridization, mRNA is selected and denatured to remove any

secondary stem-loop structures, and then electrophoresed through an agarose

gel.
⚫The mRNA is not digested with a restriction endonuclease, since these enzymes

only function on double-stranded DNA.

⚫After separating the RNA molecules by molecular weight, they are transferred

to nitrocellulose and incubated with a probe at the desired stringency.

⚫The bands that appear on the X-ray film correspond to specific mRNAs.

⚫The size of the mRNA, in nucleotides, can be calculated by the distance that the

mRNA migrated through the gel.

Western Blotting, or Immunoblotting
⚫Can be used to identify and characterize a genomic DNA fragment, cDNA, or

mRNA.

⚫A different approach needs to be employed to examine protein expression.

⚫Remember that the phenotype of an organism usually depends on the

protein.

⚫Thus, a mutant phenotype, which is usually due to a change in the DNA,

ultimately results from a change in the expression of a protein or an alteration in the

protein’s amino acid sequence.
⚫This suggests that nucleic acid hybridizations can be extremely informative for a

variety of reasons, but analyzing protein expression can be equally important.

⚫To examine the expression of a specific protein within a population of

different proteins, we also need a probe.

⚫In the case of proteins, the probe is usually an antibody, a specialized protein

that can recognize a precise, short amino- acid sequence in another protein.

⚫Antibodies can be very specific, in that each antibody may bind to only a

single protein.

⚫This procedure is called either Western blotting (a takeoff on the

Southern and Northern hybridizations) or immunoblotting.

⚫Denatured or intact proteins from a specific tissue or organism are

electrophoresed through a polyacrylamide gel rather than an agarose gel

⚫The proteins are separated by molecular weight as they move through the

gel and are then electrotransferred to nitrocellulose.

⚫The nitrocellulose is then incubated with the desired antibody, which

can bind to the specific protein on the filter.

⚫Sometimes the antibody is radioactively labeled and can then be detected

on X-ray film.
⚫Alternatively, a second antibody is covalently attached to an enzyme

(alkaline phosphatase or horseradish peroxidase), which is used in either

a colorimetric reaction to cleave a colorless compound into a colored
product or a chemiluminescent reaction
⚫This secondary antibody will specifically bind the first (primary) antibody

and the detected label would indicate the location of the desired protein.

⚫This can be used to determine the molecular weight of the desired

protein, where it is expressed in the organism, or when it is expressed

during development .

⚫This immunoblot technique can also be performed on a cDNA library. In

this case, the antibody identifies the colony that contains the cDNA that
encodes the desired protein.
 Colony or Plaque Hybridization

⚫Colony blot hybridization is applied to DNA or RNA released

from blotted microbial colonies.

⚫The microbial colonies are transferred (blotted) to a membrane.

⚫The cells are lysed in place to release the nucleic acids.

⚫ The RNA or DNA (after denaturation) is fixed to the filter and

hybridized with a labelled probe.
⚫Blocking reagent may be added prior to the probe to prevent

unspecific binding.

⚫Excess probe is washed away and the membrane is visualized by UV

or autoradiography.

⚫Colony blot hybridization can be used for screening clones or bacterial

isolates.