| Bhajan Kumar

Methods of Analysis of Nucleic Acids

Contents:
 Introduction of Nucleic Acids
 Tools and Techniques used for Nucleic Acids Analysis
 Isolation and Purification of Nucleic Acids
 Spectrophotometric Method for Nucleic Acid Quantification
 Real Time Polymerase Chain Reactions
 Gel Electrophoresis
 Nucleic Acid Hybridization
 In situ Hybridization
 Fluorescence In situ Hybridization (FISH)
 Nucleic Acid Blotting Techniques
 Nucleic Acids Sequencing Techniques
 DNA Sequencing
 Maxam-Gilbert Method of DNA Sequencing
 Sanger’s Method of DNA Sequencing
 RNA Sequencing
 Next Generation Sequencing (NGS)
 Molecular Markers
 Conclusion

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Methods of Analysis of Nucleic Acids

Methods of Analysis of Nucleic Acids

Nucleic acids are molecules which allow organisms to transfer genetic information from
generation to generation. These are of two types:
 DNA (Deoxyribonucleic Acid)
 RNA (Ribonucleic Acid)
Nucleic acids are composed of monomers called nucleotides linked together. Nucleotides
contain three parts:
 A Nitrogenous Base
 A Pentose Sugar
 A Phosphate Group
DNA
 Nitrogenous Bases: Adenine, Guanine, Cytosine, and Thymine
 Five-Carbon Sugar: 2-Deoxyribose
RNA
 Nitrogenous Bases: Adenine, Guanine, Cytosine, and Uracil
 Five-Carbon Sugar: Ribose

Fig. 1: Structure of DNA and RNA illustrated.

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Methods of Analysis of Nucleic Acids

Tools and Techniques used in Nucleic Acids Analysis

1. Isolation and Purification of Nucleic Acids:
DNA
Extraction and purification of DNA involve three basic steps:
 Breaking the cell to expose the DNA
 Removing the membrane lipids using a suitable detergent
 Precipitating the DNA using alcohol.
There are also two optional additional steps which are almost always one prior to the
precipitation step - the removal of proteins by adding a proper protease and the removal of
RNA using an RNase.
RNA
Extraction and purification of RNA involve four basic steps:
 First, disrupt the cells by adding guanidium thiocyanate and a reducing agent to the
sample and then cast it to strong shaking or rotating. This step will lyse the disulphide
bonds and deactivate the contaminant proteins present in the sample.
 After the cell breaks, add phenol and chloroform-isoamyl alcohol to isolate your RNA
sample from the solution.
 The aqueous phase contains RNA. Put it in another tube, add isopropanol and centrifugate
the solution to precipitate RNA.
 Washing the precipitate with 75% ethanol will then remove any contaminants from
sample.
It is crucial to know which RNA is less stable than DNA; so, one should only isolate it prior
to use.
DNA and RNA can also be isolated from the same biological sample by taking out a total
nucleic acid fraction and dividing it into two parts - one of which will be treated with a
DNase while the other will be treated with RNase to recover RNA and DNA, respectively.

2. Spectrophotometric Method for Nucleic Acid Quantification:

Nucleic acids are quantified to test the concentration and purity of DNA/RNA present in the
solution or mixture. It is very important because the purity of nucleic acid for use in further
submissions like PCR, restriction digestion etc. Spectrophotometric analysis is the most
commonly used technique of quantifying DNA.
Nucleic acids absorb UV light in the wavelength of 260nm, a solution containing nucleic acid
to be tested is exposed to UV light and the absorbance is measured. Greater the light
absorption, more the nucleic acid the test solution contains.

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NanoDrop:
NanoDrop is a UV-Vis Spectrophotometer device which is used to quantify nucleic acid from
micro volumes. Features of NanoDrop involves:
 Direct, easy measurements in less than 5 sec – just pipette & wipe.
 Measures DNA, RNA (A260) and Protein (A280) concentrations and the sample
purity (260/280 ratio).
 Big concentration range (2 ng/µL – 15,000 ng/µL dsDNA) without dilutions.
The modern NanoDrop series are NanoDrop 2000/2000c, NanoDrop 8000, NanoDrop Lite,
etc.
Checking the Purity of the Sample:
 The 260/280 ratio indicates the purity of the sample analysed. Pure DNA sample shows a
260/280 ratio approximately 1.8 and for pure RNA 260/280 ratio is approximately 2.

 The absorbance at 230nm is indication of other contamination; hence, the ratio of

A260/A230 is also determined. The 260/230 values for impure nucleic acid are often
greater than the corresponding 260/280 values. Expected 260/230 values are commonly in
the range of 2-2.2.
Residual chemical contamination from nucleic acid extraction procedures may result in an
overestimation of the nucleic acid concentration and/or negatively influence the
downstream analysis.

3. Real Time PCR or Quantitative PCR (qPCR):

Real-time Polymerase Chain Reactions is a laboratory technique which is used to quantity the
PCR product at the end of each amplification cycle (i.e., in real time). To do this, PCR
products are labelled during or after the primer elongation step of each amplification cycle.
Detection Methods:
a) Fluorescent Dye Based qPCR:
It involves using DNA intercalating or minor groove targeting dyes which show greater
fluorescence upon binding. With each amplification cycle, as the amount of the template
DNA rises, the number of fluorophores bound, and therefore, the intensity of the fluorescent
signal, increase. Popular fluorescent intercalating dyes are SYBR® Green I and SYBR® Gold.
Their main advantage is cost, but they are non-specific and bind all double stranded DNA and
not just the target.
b) DNA Probe Based qPCR:
It involves using fluorophore-labelled sequence-specific DNA probes. The most popular type
involves using the 5’to 3’ exonuclease activity of Taq Polymerase to chew up an

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Methods of Analysis of Nucleic Acids

oligonucleotide, which has a fluorophore and a quencher at the 5’ and 3’ ends respectively.
The exonuclease activity of the polymerase helps to separate the fluorophore from the
quencher, which results in the enhanced fluorescence.

Fig. 2: Comparison of Real Time PCR Methods.

Working of qPCR:

Fig. 3: Working Principle of Real Time PCR.

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Applications:
qPCR is used for many applications over the years. These include:
 Gene Expression (mRNA) Analysis
 microRNA and Non-Coding RNA analysis
 Genetic Variation
 Mutation Detection
 SNP Analysis
 Genotyping/Allelic Discrimination

4. Gel Electrophoresis:
Gel Electrophoresis is the physical method of separation of molecules based on their size,
charge and length.
Principle:
Electrophoresis implies the running of current through a gel containing the molecules of
interest. Depending upon their charge and size, the molecules will run through the gel in
different directions or at different speeds, allowing them to be separated from one another.
Visualization is attained by various strategies, most commonly by intercalating dyes.
Apparatus:
The gel electrophoresis apparatus consists of:
 Gel: Composed of Agar or Polyacrylamide.
 The two most common buffers for nucleic acid electrophoresis are Tris-acetate with
EDTA (TAE) and Tris-borate with EDTA (TBE), both with pH close to neutral to
favor negative charges on the nucleic acids.
 Electrophoretic Chamber: A hard plastic box or tank with a Cathode at one end and
an Anode at the opposite end.

Working Procedure:

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The gel, containing a series of wells at cathode, is placed inside the chamber and is covered
with a buffer solution. The samples are loaded into the wells with a pipette. The chamber is
connected to a power supply which applies an electric field to the solution. The electric field
causes negatively charged molecules to migrate through the gel toward the anode. DNA/RNA
molecules are negatively charged due to an abundance of Phosphate groups.
The movement of molecules is affected by porous gel matrix. The larger and denser
molecules move relatively slowly, whereas smaller, lighter molecules move more quickly.
The pores’ density and the substance type used to make gel further affect the rate of molecule
migration.
A dyed ladder or marker of known and variable molecular weight is run along the samples
which serves as a reference. The dye aids in the visualization of the marker as it moves
through the gel. Samples are also dyed for visualization. Ethidium bromide shows
fluorescence under UV light and is frequently used for crisp visualization of DNA samples.

Fig. 4: Gel electrophoresis for DNA: The gel and electrophoretic apparatus (left) and, The
separated bands of dyed DNA in the gel at the end of the experiment (right).

Applications:

Gel Electrophoresis has many applications in molecular biology and biotechnology. Some of
the important applications include:
 Separation of restriction enzyme digested DNA including genomic DNA.
 Analysis of PCR products after polymerase chain reactions to evaluate for target DNA
amplification.
 Allows the assessment of the size of DNA molecules using a DNA marker or ladder
which contains DNA fragments of different known sizes.
 Allows the rough estimation of quantity and quality of DNA.

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5. Nucleic Acid Hybridization:

Nucleic acid hybridization is a technique in which single stranded nucleic acids, DNA or
RNA, are permitted to interact; so which complexes called the hybrids are formed by
molecules with identical, complementary sequences.
Combinations:
Hybridizations are done in three combinations:
 DNA - DNA (DNA is made single stranded by heat denaturation)
 DNA - RNA
 RNA - RNA
Detection of Hybrids:
Hybrids are detected by various means:
 Visualization in the electron microscope.
 By radioactively labelling one component and removing non-complexed DNA.
 By washing or digesting with an enzyme which attacks single-stranded nucleic acids
and finally estimating the radioactivity bound.
Basic Principle:
Single-stranded DNA molecule recognizes and specifically binds to a complementary DNA
strand in a mixture of another DNA strand.
Procedure:
 Single-stranded target DNA is bound to a membrane support.
 DNA probe labelled with the detector is added.
 DNA probe pairs with the complementary targeted DNA.
 Sequence of nucleotides in the targeted DNA can be identified.

Target Probe
Membrane DNA DNA

excess
Probe DNA
Apply X-ray film

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Fig. 5: Steps of Nucleic Acid Hybridization.

Applications:
Nucleic acid hybridization is widely used:
 To test the degree of relatedness between the two DNA molecules.
 To isolate genes for cloning.
 To search for identical sequences in experimental samples of target DNA molecules.
6. In situ Hybridization (ISH):
This technique was developed by Mary-Lou Pardue and Joseph G. Gall. It is a form of
hybridization which involves using a labelled cDNA, RNA or a modified nucleic acid strand
(probe) to localize a definite DNA or RNA sequence in a portion or section of tissue (in situ)
or if the tissue is small enough (like plant seeds, Drosophila embryos) in the entire tissue, in
cells, and in circulating tumour cells (CTCs).
Significance:
In situ hybridization is used to find the position of specific nucleic acid sequences on
chromosomes or in tissues. It is a crucial step to understande the organization, regulation, and
function of genes.
Modern in-use Techniques:
The key techniques currently in use include:
 In situ hybridization to Messenger RNA with oligonucleotide and RNA probe (radio-
labelled and hapten-labelled).
 Whole Mount In situ hybridization
 Double detection of RNAs and RNA-plus protein
 Fluorescence in situ hybridization (FISH)
Here most widely used In situ hybridization technique is discussed.

7. Fluorescence In situ Hybridization (FISH):

FISH is a molecular cytogenetic technique which involves using fluorescent probe which
bind only to those fragments of a nucleic acid sequence with a high degree of sequence

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complementarity. Fluorescence Microscopy is used to locate where the fluorescent probe

binds to the chromosome.
Principles:
 DNA Probe and Target sequence are the basics of FISH.
 Before the hybridization, DNA probe is labelled indirectly with hapten (left panel) or
directly labelled through incorporating fluorophore (right panel).
 The labelled probe and target DNA are then denatured.
 Annealing of Complementary DNA sequences by combining the Denatured Probe and
Targeted DNA.
 If the probe is labelled indirectly, an extra step is needed for visualization of the non-
fluorescent hapten which involves using a detection system.
 Finally, the signals are assessed through fluorescence microscopy.

Fig. 6: Working Principle of FISH.

Applications:
FISH is used for:
 Finding specific features in DNA for use in genetic counselling, medicine, and species
identification.
 Detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells,
circulating tumour cells, and tissue samples.
 Detection of numerical and structural Chromosomal Abnormalities.
 Identification of Marker Chromosomes.
 Monitoring the effect of therapy.

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 Detection of early relapse or minimal residual diseases.

 Gene Deletion and Gene Amplification Detection.

8. Nucleic Acid Blotting Techniques:

The process of immobilization of sample of nucleic acid in solid support is called Blotting.
The blotted nucleic acids are used as a target in the hybridization experiments for detection.
Types of Blotting:
 Southern blot:
This method is usually used in molecular biology for detection of a DNA sequence in DNA
samples. The basis of this technique is DNA-DNA hybridization.

Fig. 7: Illustration of Southern Blotting.

 Northern blot:
It is implied for the detection of given RNA sequences in complex samples.

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Fig. 8: Illustration of Northern Blotting.

 Reverse Northern blot:

It differs from both Northern and Southern blotting in which DNA is first immobilized on a
blotting matrix and specific sequences are detected with labeled RNA probes.
 Western blot:
It is used for the detection of specific proteins in complex samples.

Probe with
biotinylated bait
protein
Probe with
Transfer to membrane substrate
Streptavidin
for blot detection
SA

Separate prey proteins

by electrophoresis after
cell lysis

Lagged bait protein

Directly use mini gel interacts with prey protein Erymid-conjugated streptavidin Band represents prey
for in gel detection in bands on the membrane binds and chemiluminescent protein in lysate that
or in the gel substrate produces
recordable signal
bound the labeled bait.

Fig. 8: Illustration of Western Blot immunodetection

 Colony blotting:

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Fig. 9: Procedure for Colony Blotting.

 Dot Blot:
The procedure for Dot blotting is:
 The sample (DNA or RNA) from different individuals are fragmented onto a
nitrocellulose filter in the form of dots.
 Then the DNA is denatured, and the filter is baked at 80°C to fix the DNA
strongly to the filter.
 The filter is then treated with suitable radioactive single-stranded DNA probe
under favorable hybridization conditions.
 Filter is then repeatedly washed to remove free probes.
 The hybridized probes are detected by autoradiography.

9. Nucleic Acids Sequencing Techniques:

A. DNA Sequencing:
DNA sequencing is the process to determine the order of nucleotides of given DNA
fragment. Still now, most DNA sequencing has mostly been performed using the ‘chain
termination method’ developed by Frederick Sanger.
Methods of DNA Sequencing:
 Maxam & Gilbert's Method (The Chemical cleavage)
 Fredrick Sanger's Method (Dideoxy method or Chain Termination Method)
 AUTOMATED Sequencing (dideoxy, using fluorescent tags)
 DNA-chip Sequencing

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 Sequencing of individual bases (at the atomic level)

Here two most commonly used methods have been discussed.

a) Maxam-Gilbert Method of DNA Sequencing:

It is a method of DNA sequencing which was developed by Allan Maxam and
Walter Gilbert in 1977-80. This method is primarily based on nucleobase-specific partial
chemical modification of DNA, and subsequent cleavage of the DNA backbone at sites
adjacent to the modified nucleotides.
Working Principle:
 DNA sample is separated in four groups. Each treated with a specific restriction
enzyme for A, T, G, or C.
 After cleavage, all four groups are positioned in the same apparatus for gel
electrophoresis.
This process can be automated by connecting different fluorescent dyes to the end of the
DNA fragments in each group and read off by a Scanning Detector hooking up to the
computer. This technique is used to sequence fragments.

Fig. 10: Working Principle of Maxam-Gilbert Method of DNA Sequencing.

b) Sanger’s Method of DNA Sequencing:

Sanger’s sequencing also known as “Chain Termination Method” is a methodology to
determine the nucleotides’ sequences of DNA. This method was developed by Frederick
Sanger and his colleagues; hence, named the Sanger’s Sequencing.
Working Principle:

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Sanger’s sequencing can be performed manually or, more commonly in an automated

fashion via sequencing machine. In both cases, the method goes through three basic steps,
which are described in the figure.

Fig. 10: Working Principle of Sanger’s Method of DNA Sequencing.

Applications of DNA Sequencing:

 Sequence genetic information
 Identification of mutations in genes/chromosome
 Evolutionary relationships among species
 Gene identity and mapping
 Gene expression questions

B. RNA Sequencing:
RNA is comparatively less stable in the cell, and more is susceptible to nuclease attack
experimentally. As RNA is generated through transcription from DNA, the information is
already available in the cell’s DNA. However, it is sometimes necessary to sequence RNA.
The sequence of DNA gives genetic profile of an organism, while sequencing of RNA shows
only the sequences which are actively expressed in the cells.
Working Principle:
To sequence RNA, the usual method is first to reverse transcribe the RNA extracted from the
sample to generate cDNA fragments. This can then be sequenced.

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Fig. 10: RNA Sequencing

Workflow.

10. Next Generation Sequencing (NGS)

The next generation sequencing is a most advanced sequencing technique presently denoted
by a variety of applicable platforms. This immensely parallel sequencing is a highly
complicated procedure which is capable of sequencing millions of dissimilar short DNA
fragments in a single cycle.
Technical Principle:
The basic principle is NGS platform-specific, but they all offer enormous amount of data
which must be assessed by a good bioinformatical software or Linux pipelines.

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Generic Overview of NGS

Input DNA
Fragmentation
Library Preparation
Fraumeni library

Clonal amplmeMon of each from Fraumeni

Clonal Amplification

Emulsion Bridge
Pen Amplification

Sequencing
Generation of luminescent in fluorescent images

Sequence

Fig. 11: Overview of Next Generation Sequencing Workflow.

Significance of NGS:
These techniques provide the most comprehensive picture of the species genome.

11. Molecular Markers:

In genetics, a molecular marker (commonly known as Genetic marker) is a portion of
DNA which is associated with a location in the genome. Molecular markers are widely used
in molecular biology and Biotechnology to find a sequence of DNA in a pool of unknown
DNA.

Sr. No. List of Important Markers Acronym

1 Restriction Fragment Length Polymorphism RFLP

2 Random Amplified Polymorphic DNA RAPD

3 Amplified Fragment Length Polymorphism AFLP

4 Variable Number Tandem Repeat VNTR

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5 Oligonucleotide Polymorphism OP

6 Random Amplified Polymorphic DNA RAPD

7 Single Nucleotide Polymorphism SNP

8 Allele Specific Associated Primers ASAP

9 Inverse Sequence-tagged Repeats ISTR

10 Inter-retrotransposon Amplified
IRAP
Polymorphism

Characteristics of Important Genetic Markers:

CHARACTERISTICS RAPD RFLP AFLP

BASIC PRINCIPLE DNA amplification Restriction DNA amplification

Digestion

DETECTION DNA staining Southern blotting DNA staining

TECHNIQUE

PRIMER Yes (random None Yes (selective

REQUIREMENTS primer) primer)

PROBE None set of specific None

REQUIREMENTS probes

DOMINANT/ CO- Dominant Co dominant Dominant (Co-

DOMINANT dominant)

Conclusive Remarks:
There are numerous techniques for the analysis of nucleic acids depending upon the purpose
of applications. The most important and commonly in-use techniques have been discussed in
this article. They have vast application in many fields of sciences and medicine.
Nucleic acids are an important class of biomolecules. They are unit of inheritance and
regulators of all the essential activities in the living systems. So, their physiological study and
quantitative analysis is of huge significance.

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