Chapter 3

Nucleic acid hybridization and amplification

Learning out comes
At the end of this chapter the students will be able to
know:
 Nucleic acid hybridization
 Probe and Target Sequences
 Methods of Nucleic Acid hybridization
 Polymerase Chain Reaction (PCR) and its
Applications
 Variations in PCR and their Applications
 What is nucleic acid hybridization?
Nucleic acid hybridization is the formation of a
stable duplex between two complementary strands
of nucleic acid by means of hydrogen bonding
between base pairs.
It is a process used to identify specific DNA
sequences.
Specific DNA probes are denatured and annealed to
sample DNA that has also been denatured.
• Probes used in hybridization reactions are usually
chemically synthesized DNA or RNA that has been
labeled with a fluorescent dye or radioactive
isotope such as 32P.
The techniques of nucleic acid hybridization
is established and developed on the basis of the
denaturation and renaturation of nucleic
acids.
Hydrogen bonds in double stranded nucleic
acids can be disrupted by some
physicochemical elements and two strands of
nucleic acids are separated in to single strand.
Nucleic acid hybridization as a technique
involves using a labeled nucleic acid probe,
which is a known DNA or RNA fragments ,
to bind with the target nucleic acids.
 Probe form
• A probe is normally a short sequence of nucleotide
bases that will bind to specific regions of a target
sequence of nucleotides.
• The degree of homology between target and probe
results in stable hybridization.
• In developing a probe, a sequence of nucleotides
must be identified, reproduced in sufficient quantity,
and tagged with a compound (label) that can be
detected.
• An ideal probe is single-stranded nucleic acid that
can hybridize to the target of concern.
• The probe is composed of either DNA or RNA, with
Probe labeled with detectable tracer is the
prerequisite for determining a specific DNA
sequence or a gene in a sample or genomic DNA by
nucleic acid hybridization.
The target nucleic acids to be analyzed are usually
denatured and then mixed with the labeled probe in
the hybridization system.
The probe will bind to the segment nucleic acid
with complementary sequence under proper
conditions.
The hybridization can be identified by the detection
of the tracer labeling the probe.
Then the existence of specific gene can be
 Hybridization probes
It is a nucleic acid fragment that is complementary to
another nucleic acid sequence and thus , when labeled
(with radioisotopes , fluorescent dye ,ets) can be used to
identify complementary segments.

A probe actually hybridizes to single stranded nucleic acid

( DNA/RNA )molecules because of complementary
between the probe and target.

Nucleic acid probes can be synthesized in the laboratory, as

single and double stranded probes, but the working nucleic
acid should be a single stranded only to bind with
complementary target sequence.
 Probe size
 Probes can range in size from as short as 10 nucleotide bases
(molecular weight of 3,300) to as long as 10,000 bases or more
(molecular weight of 3,300,000).
 The most common size range for most probes is between 14 and 40
bases.
 Short probes tend to hybridize nucleic acids at very high rates (in
minutes), whereas longer probes may require reaction times of hours
to achieve a stable hybridization.
 However, short probes do have some disadvantages.
 They are subject to more nonspecific hybridizations, limited in
specificity, and more difficult to label.
 Long probes hybridize more stable than short probes at high
temperatures and low salt concentrations .
 The advantages of rapid hybridization, longer stability, ease of
preparation, and the ability to detect minor changes in the target
nucleic acid by the shorter probes favor their use in most commercial
 Specificity of the probe
 The base sequence of the probe and the conditions under which
the probe is used determine its specificity.
 It is not necessary to know the function of the target nucleic acid
or even the identity of the target or probe sequence before a
probe can be used.
 The only requirements are that the probe hybridizes specifically to
the target nucleic acids and that the target nucleic acids be
unique.
 The probe must not hybridize to itself or to non-target nucleic
acids in either the indigenous microbial flora or the clinical
specimen.
 To be useful, probes must be detectable after hybridization with
the target.
 Labels can be either attached or incorporated into the probe
• Stages of hybridization in nucleic acids
• The hybridization of a radioactive probe to filter bound DNA or RNA is
one of the most informative experiments that is performed in
molecular genetics.
• There are two different types of nucleic acid hybridization techniques
generally used , which are called Southern blotting and northern
blotting techniques.
• The term blotting refers to the transfer of biological samples from a
gel to a membrane and their subsequent detection on the surface of a
membrane.
• Southern blot is a method used in molecular biology for detection of
specific DNA sequence in a DNA samples.
• Southern blotting combines transfer of electrophoresis-separated
DNA fragments to a filter membrane and subsequent fragment
detection by probe hybridization.
• The method is named after its inventor, the British biologist Edwin
Mellor Southern.
Steps describe the Southern transfer procedure
1. Digest DNA with the restriction enzyme of choice.
2. Load the digestion onto a agarose gel and apply an electrical
current. DNA is negatively charged so it migrates toward the "+"
pole. The distance a specific fragment migrates is inversely
proportional to the fragment size.
3. Stain the gel with EtBr, a fluorescent dye which intercalates
into the DNA molecule. The DNA can be visualized with a UV
light source to assess the completeness of the digestion.
4. Denature the double-stranded fragments by soaking the gel in
alkali (>0.4 M NaOH).
5. Transfer the DNA to a filter membrane (nylon or nitrocellulose)
by capillary action. Typically a Southern transfer setup contains
buffer, sponge, filter paper, the gel containing the nucleic acid,
a nylon or nitrocellulose membrane, more filter paper and
paper towels to catch the buffer that passed through all of the
 Steps involved in southern blotting
1. Extract and purify DNA
from the cells
2.DNA is restricted with
enzymes
3.Denature DNA
4.Separated by
electrophoresis
5.Transfer to nitro cellulose
paper
6.Add labeled probes for
hybridization to take place.
7.Wash off unbound probe
8.Autoradiograph.
Applications
• To identify specific DNA fragment in DNA sample
• To isolate desired DNA for construction of rDNA
• Identify mutations, deletions ,and gene
rearrangements
• Used in prognosis of cancer and in prenatal
diagnosis of genetic diseases.
• Diagnosis of infectious disease
• In DNA finger printing
• Paternity and maternity testing
• Criminal identification and forensics
• Personal identifications.
 Northern blotting
• Northern blotting is a technique for detection of specific
RNA sequences.
• Northern blotting was developed by James Alwine and
George Starkat at stanford univesity (1979) and was
named such by analogy to southern blotting.
• Northern blots are used to determine the identity, size,
and abundance of specific RNA sequences.
• Northern blot protocols begin with RNA isolation, and
separation techniques vary depending on RNA size.
• Large RNAs are separated by electrophoresis on a
formaldehyde agarose gel or glyoxal agarose gel, which
prevents normal base paring and maintains RNA in a
denatured state.
• Small RNAs are separated on a denaturing
polyacrylamide gel.
• The RNA is then transferred from the gel to a
nylon membrane which is then incubated with
a radioactively or non-isotopically labelled
probe.
• The unhybridized probe is removed by
washing with buffer.
• Radiolabelled probes are visualized with X-ray
film, and enzymatically labelled probes are
visualized with chemiluminescence.
Applications of Northern Blot Technique
The technique can be used for the identification and
separation of RNA fragments collected from
different biological sources.
 Northern blotting is used as a sensitive test for the
detection of transcription of DNA fragments that
are to be used as a probe in Southern Blotting.
 It also allows the detection and quantification of
specific mRNAs from different tissues and different
living organisms.
Northern blotting is used as a tool for gene
expression studies related to over expression
of cancer-causing genes, and gene expression
during transplant rejects.
Northern blotting has been used as a
molecular tool for the diagnosis of diseases
The process is used as a method for the
detection of viral microRNAs that play
important roles in viral infection.
• Another type of blotting technique is called Western blotting,
which uses the same principles of hybridization but is used for
identifying specific protein molecules using protein probes
labeled with a fluorescent dye or with a radioisotope such as
35S.
• The most widely used protein probes for Western blots are
antibodies, either monoclonal or polyclonal.
• These are used to detect target proteins called antigens.
• A combination of Southern blot and Northern blot techniques
is also used in the characterization of specific DNA-binding
proteins and this technique is called a Southwestern blot, since
it involves hybridization of both protein and DNA.
• Short regions of target DNA sequences are labeled and serve
as probes for hybridization reactions.
• Many DNA-binding proteins can be characterized by this
method
 POLYMERASE CHAIN REACTION(PCR)
Generate multiple
copies of a particular
DNA sequence
 invented by Kary
Mullis (1983)
 won Nobel prize in
1993
It is called
“polymerase” because
the enzyme used is DNA
polymerases
It is called “chain”
because the products of
the first reaction become
substrates of the next
 ESSENTIAL COMPONENTS OF PCR
• The following are the essential
components of PCR
 Thermal cyclers
 Target DNA
 Two primers
 Thermostable DNA polymerase
 Buffers
 Deoxynucleotide triphosphates (d
NTP’s)
 Monovalent \bivalent cation
 Water
 Relies on thermal cycling
 ‘Thermocycler’

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Target DNA and nucleotides
• It consists of the DNA to be amplified.
• The segment represents a small part of a large
and complex mixture of a specific DNA of a
genome. The shape of DNA is a double helical
structure which consists of nucleotides that
wind around each other in a helical shape.
• PCR requires a template molecule (i.e.) DNA\
RNA.
• The 4 nucleotide components are like building
blocks used to construct genome molecules.
• The nucleotide bases are adenine, thymine,
cytosine and guanine which also needs a small 23
Two primers
• Primers are short, artificial DNA strands not more
than 50 nucleotides, which determines the
beginning and the end of the region to be amplified,
the polymerase synthesizes the complementary
sequence from each primer.

• Primers limit the DNA sequence to be replicated

and results in the amplification of a particular DNA
sequence.

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• If template contains A nucleotide, enzyme adds on T
nucleotide to the primer and if template contains G
nucleotide, enzyme adds on C nucleotide to the primer.
• Two components that are considered for a primer are
length of the primer and actual sequence of the primer.
The primers depend upon
• Primer length
• Annealing and melting temperature
• Specificity
• Complementary primer sequences
 The length of the primer should be as short as possible.
 The annealing temperature should below at least 5 C
than the melting point temperature
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 Thermostable DNA polymerase
• Taq obtained from Thermus aquaticus are available
commercially to serve as a standard reagent for PCR
reaction.
• The DNA polymerase from Thermus Aquaticus is
stable at 95’C.
• Depending on the ability, fidelity, efficiency to
synthesize large DNA products, a wide choice of
enzymes is now available.
• For routine PCRs, Taq polymerase (0.5-2.5 units per
standard 25-50 µL reaction) remains the enzyme of
choice.
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 PCR stages
 Three main stages or steps
1. Denaturation
 heating the reaction to 94–98 °C
 breaks the base pairs and releases ssDNA as templates

Heat

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2. hybridization or annealing
o Temperature lowered to 50–65 °C for 20–60 sec
o 2-5 oC below the Tm of primers
o Two primers are supplied in molar excess
o Primers bind to the complementary region and
anneal to ssDNA

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3. Extension/ elongation
o DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand
o 72–80 °C, commonly a temperature of 72 °C is used

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o 28-30 cycles are usually sufficient in a reaction
o Little quantitative changes was noticed when increasing
the number of cycles up to 60

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Studying PCR Products
• PCR is often the starting point for a longer series
of experiments in which the amplification
product is studied in various ways in order to
gain information about the DNA molecule that
acted as the original template.
• Although a wide range of procedures have been
devised for studying PCR products, three
techniques are particularly important and they
are-
• Gel electrophoresis of PCR products
• Cloning of PCR products
• Sequencing of PCR products.
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 The variants of PCR
Multiplex PCR :
 is a widely used molecular biology technique for
amplifying multiple targets in a single PCR
experiment.
 In a multiplexing test, more than one target
sequence can be amplified using multiple initiator
pairs in a reaction mixture.
 As an extension of the practical use of PCR, this
technique has the potential to achieve significant
savings in time and effort within the laboratory
without compromising the usefulness of the
experiment. 32
• Types of Multiplex PCR
• Multiplication reactions can be broadly divided into two
categories:
1. One template PCR reaction
 This technique uses a single template that can be genomic
DNA with multiple pairs of forward and reverse primers to
amplify specific regions within the template.
2. Multiple PCR template reaction
• Uses multiple templates and several primer sets in the
same reaction tube.
• The presence of multiple DNA template may cross-hybridize
with each other and potentially mislead with other DNA
template.
• Multiple PCR reaction to amplify multiple target sequences
in a single reaction tube,
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Fig 1: Multiplex PCR

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Quantitative/Real-Time (q) PCR:
 Is specialized techniques that allows the PCR reactions to be
visualized in a ’real - time’ as the reaction progresses.
 This enables researchers to quantify the amount of DNA in
the sample at the start of reaction.

 This technique is used to measure the amount of target DNA

present while simultaneous amplifying it.
• To do this, non-specific fluorescent dyes or DNA
oligonucleotide fluorescent probes are used.

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 “real-time” PCR (Q-PCR)
 The recent development of “real-time” PCR (Q-
PCR) added great advantages to traditional PCR.

 What is the difference between PCR and real-time PCR?

 Conventional PCR is used to detect the presence or

absence of certain genomic fragments,

 Whereas Real-Time PCR is used to detect the

expression level of that fragment in the organism.
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• Real time PCR has become cornerstone of
molecular biology:
Gene expression analysis
 Cancer research
 Drug research
Disease diagnosis
Viral quantification
Food testing
Transgenic research

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Colony PCR
• Small quantities of bacterial cells from bacterial
colonies are added to the PCR mix. An initial
extended annealing period or a shortened
denaturation step at 100°C is used to release the
DNA.
• This can be used to screen for correct DNA vector
constructs.
• Vector primers can also be used.
• Target DNA fragments are first inserted into a cloning
vector and a single set of primers are designed for
the areas of the vector flanking the insertion site,
resulting in amplification of the inserted sequence.
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• Allele-specific PCR: Rather than designing
primers for an invariant part of the genome in
order to amplify a more polymorphic area
between them, at least one of the primers used in
this variation of PCR is complementary to a
polymorphic area, with mutations located at its
3’ end.
 Since mismatched primers will not initiate
replication whereas matched primers will,
amplification is indicative of the mutation being
present.
 This is used in SNP genotyping.
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 Applications of PCR:
1. Identification and analysis of mutations in
eukaryotic DNA
 DNA deletions and insertions can be detected by a
change in the size of the PCR product.
 Failure to produce any PCR fragment indicates that
the primer is found in an area of deletion.
Mutations can be identified by hybridizing PCR-
generated DNA fragments to radioactively labelled
RNA probes and digesting the DNA-RNA complexes
with RNase A. Digestion will occur if the complex
contains any mismatches.
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 Localization of mutations has also been achieved by
examining PCR products using denaturing gradient
gel electrophoresis (DGGE).This separates about 50%
of DNA strands o1000 bp in length that have single
base changes.
2. Detection of amplified oncogenes - differential PCR
 The oncogene of interest and a single-copy reference
gene are co-amplified in the same PCR tube.
 Gene amplification is determined by comparing the
intensity of the oncogene PCR product with that of
the amplified reference single-copy gene.
3. Detection of pathogenic organisms in clinical samples
4. Identif|cation of biological and forensic samples

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5. Gene polymorphisms - RFLPs, STRPs
6. Gene expression-
Growth factors during wound healing.
Gene expression during embryogenesis.
 Quantitation of gene expression in various
organs, e.g. dystrophin
7.cDNA cloning
8. Genomic DNA cloning
9. DNA sequencing.

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