This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.

1. Nucleic acid Hybridization
Dr.N.C.J.Packia Lekshmi
NICHE

2. Introduction
• DNA–DNA hybridization generally refers to a molecular biology
technique that measures the degree of genetic similarity between
pools of DNA sequences.
• It is usually used to determine the genetic distance between two
organisms.
• This has been used extensively in phylogeny and taxonomy.
• In order to compare DNA from different species, scientists use a
technique called DNA hybridization.

3. • Nucleic acid hybridization allows scientists to compare and analyze
DNA and RNA molecules of identical or related sequences.
• In a hybridization experiment, the experimenter allows DNA or RNA
strands to form Watson‐Crick base pairs.
• Sequences that are closely related form base‐paired double helices
readily; they are said to be complementary.
• The amount of sequence complementarity is a measure of how
closely the information of two nucleic acids relate.
• The complementary strands can be both DNAs, both RNAs, or one
of each.

5. Principle
• DNA is double stranded, and individual bases bind based on Chargaff's rules
of complementary bases: adenine pairs with thymine and cytosine pairs with
guanine.
• When DNA is heated, the hydrogen bonds holding the base pairs together
dissolve and the DNA separates into two single strands during a process
known as DNA melting.
• The temperature at which the DNA strands separate is called the melting
temperature. When DNA is cooled, complementary bases realign and bind
through hydrogen bonding.
• During DNA-DNA hybridization, the DNA of each species is heated to the
melting temperature to produce single strands.
• Then, a strand of DNA from each species is combined and allowed to anneal
together (recombine).
• Then the sample is gradually heated. If the species DNA is very similar, they
will form many hydrogen bonds through the base pairs and the melting
temperature will need to be higher to separate the strands.
• If the species DNA is dissimilar, meaning the species are unrelated, the
melting temperature will be lower because there will be fewer hydrogen bonds
to break.

7. Procedure
• Single stranded target DNA is bound to a membrane
support.
• DNA probe labeled with detector substance is added.
• DNA probe pairs with the complementary target DNA.
• Sequence of nucleotide in the target DNA can be
identified.

8. • A DNA probe or a gene probe is a synthetic, single-stranded DNA molecule
that can recognize and specifically bind to a target DNA (by complementary
base pairing) in a mixture of biomolecules.
• DNA probes are either long (> 100 nucleotides) or short (< 50 nucleotides),
and may bind to the total or a small portion of the target DNA.
• There is a wide variation in the size of DNA probes used (may range from
10 bases to 10,000 bases).
• The most important requirement is their specific and stable binding with
target DNAs.

9. DNA Probes synthesis
• A great majority of DNA probes are chemically synthesized in the
laboratory.
• There are, however, many other ways of obtaining them-isolation of
selected regions of genes, cloning of intact genes, producing from
mRNAs.
Synthesis of DNA probes from mRNA:
• The mRNA molecules specific to a particular DNA sequence
(encoding a protein) are isolated. By using the enzyme reverse
transcriptase, complementary DNA (cDNA) molecules are
synthesized. This cDNA can be used as a probe to detect the target
DNA.
Isolation of selected regions of genes:
• The DNA from an organism (say a pathogen) can be cut by using
restriction endonucleases. These DNA fragments are cloned in
vectors and the DNA probes can be selected by screening.

10. Mechanism of Action of DNA Probes:
• The basic principle of DNA probes is based on the
denaturation and renaturation (hybridization) of DNA.
• When a double-stranded DNA molecule is subjected to
physical (temperature > 95°C or pH < 10.5) or
chemical (addition of urea or formaldehyde) changes,
the hydrogen bonds break and the complementary
strands get separated. This process is called
denaturation.
• Under suitable conditions (i.e., temperature, pH, salt
concentration), the two separated single DNA strands
can reassemble to form the original double-stranded
DNA, and this phenomenon is referred to as
renaturation or hybridization.

11. Detector system in DNA Hybridization
• Radioactive detector system
• Non-Radioactive detector system
Radioactive Detection System:
• The DNA probe is usually tagged with a radioactive isotope
(commonly phosphorus-32).
• The target DNA is purified and denatured, and mixed with
DNA probe.
• The isotope labeled DNA molecules specifically hybridizes
with the target DNA.
• The non-hybridized probe DNA is washed away.
• The presence of radioactivity in the hybridized DNA can be
detected by autoradiography.
• This reveals the presence of any bound (hybridized) probe
molecules and thus the complementary DNA sequences in
the target DNA.

12. Non-radioactive Detection System:
• The disadvantage with the use of radioactive label is that the isotopes have
short half-lives and involve risks in handling, besides requiring special
laboratory equipment.
• So, non-radioactive detection systems (e.g., biotinylation) have also been
developed. Biotin-labeled (biotinylated) nucleotides are incorporated into the
DNA probe.
• The detection system is based on the enzymatic conversion of a chromogenic
(colour producing) or chemiluminescent (light emitting) substrates.
• The procedure commonly adopted for chemiluminescent detection of target
DNA.
• A biotin labeled DNA probe is hybridized to the target DNA. The egg white
protein avidin or its bacterial analog streptavidin is added to bind to biotin.
• Now a biotin labeled enzyme, such as alkaline phosphatase is added which
attaches to avidin or streptavidin. These proteins have four separate biotin-
binding sites.
• Thus, a single molecule (avidin or streptavidin) can bind to biotin-labeled DNA
probe as well as biotin-labeled enzyme.
• On the addition of a chemiluminescent substrate, the enzyme alkaline
phosphatase acts and converts it to a light emitting product which can be
measured.

13. • The biotin-labeled DNA is quite
stable at room temperature for
about one year.
• The detection devices using
chemiluminescence are preferred,
since they are as sensitive as
radioisotope detection, and more
sensitive than the use of
chromogenic detection systems.

14. PCR in the use of DNA probes:
• DNA probes can be successfully used for the identification of target DNAs from
various samples — blood, urine, feces, tissues, throat washings without much
purification.
• The detection of target sequence becomes quite difficult if the quantity of DNA is very
low.
• In such a case, the polymerase chain reaction (PCR) is first employed to amplify the
minute quantities of target DNA and identified by a DNA probe.

15. DNA Probes and Signal Amplification:
• Signal amplification is an alternative to PCR for the identification of minute quantities
of DNA by using DNA probes.
• In case of PCR, target DNA is amplified, while in signal amplification it is the target
DNA bound to DNA probe that is amplified.
There are two general methods to achieve signal amplification.
1. Separate the DNA target—DNA probe complex from the rest of the DNA molecules,
and then amplify it.
2. 2. Amplify the DNA probe (bound to target DNA) by using a second probe. The RNA
complementary to the DNA probe can serve as the second probe. The RNA-DNA-
DNA complex can be separated and amplified. The enzyme O-beta replicase which
catalyses RNA replication is commonly used.

16. Fluorescence In Situ Hybridization
• Fluorescence in situ hybridization (FISH) is a laboratory
method used to detect and locate a DNA sequence, often on
a particular chromosome.
• In the 1960s, researchers Joseph Gall and Mary Lou Pardue
found that molecular hybridization could be used to identify
the position of DNA sequences in situ (i.e., in their natural
positions within a chromosome).
• In 1969, the two scientists published a paper demonstrating
that radioactive copies of a ribosomal DNA sequence could
be used to detect complementary DNA sequences in the
nucleus of a frog egg.
• Since those original observations, many refinements have
increased the versatility and sensitivity of the procedure to
the extent that in situ hybridization is now considered an
essential tool in cytogenetics.

17. Principle of FISH
• Fluorescence in situ hybridization (FISH) is a technique that uses
fluorescent probes which bind to special sites of the chromosome
with a high degree of sequence complementarity to the probes.
• The fluorescent probes are nucleic acid labeled with fluorescent
groups and can bind to specific DNA/RNA sequences.
• Thus, we can understand where and when a specific DNA
sequences exist in cells by detecting the fluorescent group.
• It was developed in the early 1980s.
• Fluorescence microscopy can be used to find out where the
fluorescent probe is bound to the chromosomes and flow cytometry
can be used to detect the binding quantitatively.
• This FISH protocol is for a Cy5 and FAM labeled probe used in flow
cytometry detection and fluorescence microscopy detection.

19. Application
An ideal technique for identification of:
• infectious diseases
• cancer
• genetic disorders
• mitochondrial disorders
• an aid to personalized and precision medicine based on the
knowledge of pharmacogenomics.

20. Nucleic acid hybridization assay
• A hybridization assay comprises any form of quantifiable
hybridization i.e. the quantitative annealing of two complementary
strands of nucleic acids, known as nucleic acid hybridization.
• Hybridization assays involve labelled nucleic acid probes to identify
related DNA or RNA molecules (i.e. with significantly high degree of
sequence similarity) within a complex mixture of unlabelled nucleic
acid molecules.
• Antisense therapy, siRNA, and other oligonucleotide and nucleic
acid based biotherapeutics can be quantified with hybridization
assays.
• Signalling of hybridization methods can be performed using
oligonucleotide probes modified in-synthesis with haptens and small
molecule ligands which act homologous to the capture and detection
antibodies.
• As with traditional ELISA, conjugates to horse radish peroxidase
(HRP) or alkaline phosphatase (AP) can be used as secondary
antibodies.

21. Sandwich hybridization assay
• In the sandwich hybridization
ELISA assay format, the antigen
ligand and antibodies in ELISA are
replaced with a nucleic acid
analyte, complementary
oligonucleotide capture and
detection probes.
• Generally, in the case of nucleic
acid hybridization, monovalent salt
concentration and temperature are
controlled for hybridization and
wash stringency, contrary to a
traditional ELISA, where the salt
concentration will usually be fixed
for the binding and wash steps (i.e.
PBS or TBS).
• Thus, optimal salt concentration in
hybridization assays varies
dependent upon the length and
base composition of the analyte,
capture and detection probes.

22. Competitive hybridization assay
• The competitive hybridization assay is similar to a traditional competitive
immunoassay.
• Like other hybridization assays, it relies on complementarity, where the
capture probe competes between the analyte and the tracer–a labelled
oligonucleotide analog to the analyte.

23. Hybridization-ligation assay
• In the hybridization-ligation assay a template probe replaces the capture probe in the sandwich
assay for immobilization to the solid support.
• The template probe is fully complementary to the oligonucleotide analyte and is intended to serve
as a substrate for T4 DNA ligase-mediated ligation.
• The template probe has in addition an additional stretch complementary to a ligation probe so that
the ligation probe will ligate onto the 3'-end of the analyte.
• Albeit generic, the ligation probe is similar to a detection probe in that it is labelled with, for
example, digoxigenin for downstream signalling. Stringent, low/no salt wash will remove un-
ligated products.
• The ligation of the analyte to the ligation probe makes the method specific for the 3'-end of the
analyte, ligation by T4 DNA ligase being much less efficient over a bulge loop, which would
happen for a 3' metabolite N-1 version of the analyte, for example.
• The specificity of the hybridization-ligation assay for ligation at the 3'-end is particularly relevant
because the predominant nucleases in blood are 3' to 5' exonucleases.
• One limitation of the method is that it requires a free 3'-end hydroxyl which may not be available
when targeting moieties are attached to the 3'-end, for example.
• Further, more exotic nucleic acid chemistries with oligonucleotide drugs may impact upon the
activity of the ligase, which needs to be determined on a case-by-case basis.

24. Dual ligation hybridization assay
• The dual ligation hybridization assay (DLA)
extends the specificity of the hybridization-
ligation assay to a specific method for the parent
compound.
• Despite hybridization-ligation assay's robustness,
sensitivity and added specificity for the 3'-end of
the oligonculeotide analyte, the hybridization-
ligation assay is not specific for the 5' end of the
analyte.
• The DLA is intended to quantify the full-length,
parent oligonucleotide compound only, with both
intact 5' and 3' ends. DLA probes are ligated at
the 5' and 3' ends of the analyte by the joint
action of T4 DNA ligase and T4 polynucleotide
kinase.
• The kinase phosphorylates the 5'-end of the
analyte and the ligase will join the capture probe
to the analyte to the detection probe.
• The capture and detection probes in the DLA can
thus be termed ligation probes. As for the
hybridization-ligation assay, the DLA is specific
for the parent compound because the efficiency
of ligation over a bulge loop is low, and thus the
DLA detects the full-length analyte with both
intact 5' and 3'-ends.
• The DLA can also be used for the determination
of individual metabolites in biological matrices.

25. Nuclease hybridization assay
• The nuclease hybridization assay, also
called S1 nuclease cutting assay, is a
nuclease protection assay-based
hybridization ELISA.
• The assay is using S1 nuclease, which
degrades single-stranded DNA and RNA
into oligo- or mononucleotides, leaving
intact double-stranded DNA and RNA.
• In the nuclease hybridization assay, the
oligonucleotide analyte is captured onto the
solid support such as a 96-well plate via a
fully complementary cutting probe.
• After enzymatic processing by S1 nuclease,
the free cutting probe and the cutting probe
hybridized to metabolites, i.e. shortmers of
the analyte are degraded, allowing signal to
be generated only from the full-length
cutting probe-analyte duplex.
• The assay is well tolerant to diverse
chemistries, as exemplified by the
development of a nuclease assay for
morpholino oligonucleotides.
• This assay set-up can lack robustness and
is not suitable for validation following the
FDA's guidelines for bioanalytical method
validation