Int. J. LifeSc. Bt & Pharm. Res.

2013 K Vasavirama, 2013

ISSN 2250-3137 www.ijlbpr.com

Vol. 2, No. 2, April 2013
© 2013 IJLBPR. All Rights Reserved
Review Article

MOLECULAR PROBES
AND THEIR APPLICATIONS
K Vasavirama1*

*Corresponding Author: K Vasavirama,  vasavi8@gmail.com

Molecular probes are small DNA or RNA segments that recognize complementary sequences
in DNA or RNA molecules that allow identification and isolation of these specific sequences
from an organism. Extraordinary advances in human molecular genetics have occurred over
the past decade which has revolutionized the entire scenario of biological sciences. One
component of those advances is the increase in the use of molecular probes. These probes
serve as the resources for a variety of applications. These molecular markers have acted as
versatile tools and have found their own position in various fields like taxonomy, physiology,
embryology and genetic engineering.

Keywords: DNA probe, RNA probe, cDNA probe, RFLP, DNA fingerprinting

INTRODUCTION Although, initially these probes were developed

and used for genetic engineering research but
A stretch of DNA or RNA sequence that can detect
a target sequence in the genome is called as are now frequently used for a variety of purposes
probe. Ever since their development, they are including diagnosis of infectious diseases
constantly being modified to enhance their utility (Katoch et al., 1994,1997; Kaminski et al., 1995;
and to bring about automation in the process of Sharma et al., 1996; Belak et al., 2009;Bexfield
genome analysis. The discovery of PCR (Mullis et al., 2011; Palacios et al., 2009), identification
et al., 1986; Saiki et al., 1988) was a landmark in of food contaminants (Zhang et al., 2012; Goji et
this effort and proved to be a unique process that al., 2012), variety of microbiological tests (Chuba
brought about a new class of DNA profiling et al., 1998;Smorawinska et al., 1992) and
markers. This facilitated the development of forensic tests. Probes can also be used to identify
marker-based gene tags, map-based cloning of different varieties of crop species (Cordeiro et al.,
agronomically important genes, variability studies, 2001; Anderson et al., 1993; Nagaraju et al.,
phylogenetic analysis, marker-assisted selection
2002). For basic studies in molecular biology
of desirable genotypes etc.
laboratories these are frequently used for

1
Department of Biotechnology, Gitam Institute of technology, Gitam University, Visakhapatnam – 530045.

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

identification and isolation of genes or related fragments of different sizes. Isolate DNA of
sequences. specific fragment from a particular band identified
through southern blots by hybridization with
In theory any nucleic acid can be used as a
specific labeled mRNA or cDNA molecules. Clone
probe provided it can be labeled to permit
this DNA in a vector. Allow chimeric vector to infect
identification and quantitation of the hybrid
bacteria for multiplication where it can make
molecules formed between the probe and
billions of copies. DNA probes prepared in this
sequence to be identified. In practice, double and
manner can be used for southern blotting and
single standard DNAs, mRNAs, and other RNAs
RFLP analysis.
synthesized in vitro are all used as probes. DNA/
RNA probe assays are faster and sensitive so RNA Probes
that many conventional diagnostic tests for High specific activity RNA probes or riboprobes
viruses and bacteria involving culturing of the may also be synthesized from DNA templates
organisms are being fast replaced by molecular cloned in expression vectors such as SP6 (which
probe assays. While culture tests can take days infects Salmonella typhimurium) and T7 phage
or even months, molecular probe assays can be (infects E.coli). This is achieved through RNA
performed with in few hours or minutes. synthesized in vitro and labeled simultaneously
Molecular probes can be broadly categorized with labeled nucleotides.
into DNA probes and RNA probes, sometimes Usually SP6 and T7 systems can be and have
cDNA probes and synthetic oligonucleotide been utilized to express whole RNAs, but for
probes can also be used for various purposes. making a probe, only a short labeled RNA is
The use of molecular probes has become sufficient. To enable such probes to be transcribed
todays most sophisticated and sensitive into uniform lengths, it is practice to linearize the
technology for a variety of uses involving plasmid by cleaving it with a restriction enzyme.
biological systems both in basic and applied The vector thus carries the input in the following
studies in the field of molecular biology and order phage promoter-Enzyme 1- Enzyme 2. The
biotechnology including their commercial use. template DNA is inserted at the number 1 site
and composite is treated with restriction enzyme
Preparation of Probes 2. So now this creates a linear DNA with a
Different types of probes can be prepared in promoter, the template DNA and automatic
various ways. termination site at the end cleaved with enzyme
DNA Probes 2. The mRNAs fall of when they reach this end of
Extract the DNA from an animal or plant the vector. Such templates reflect to run of
tissue.Digest extracted DNA with a restriction templates and are uniform in size and are easier
enzyme such as EcoRI or Hind III which cuts DNA to isolate from the reaction mixture. RNA probes
at specific sites or positions where a specific prepared in this manner can be used for northern
sequences recognized by the enzyme is found. blotting and in situ hybridization.
Run the digested DNA on an agarose or RNA probes offer several advantages over
polyacrylmide gel electrophoresis to separate DNA probes. Since these are single stranded and

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

provide improved signal or hybridization blots. End labeling

There is lack of competition of probe/ probe In this technique probe is isolated and end labeled
hybridization. Even though, some advantages are by removing the 5'-terminal phosphate using
there wide spread presence of ribonuclease alkaline phosphatase first and adding a
creates some problems in their preparation and 32
P-labeled phosphate with the help of a kinase.
use. So RNA probes are more sensitive to End labeled probes are far less labeled than the
degradation than equivalent DNA probes, transcribed ones with labels at several
therefore extreme care must be taken in the nucleotides in the strand.
preparation of RNA probes by keeping all glass
Nick Translation
ware free of ribonuclease.
It is one of the commonly used techniques for
cDNA Probes producing a radioactive probe. A purified phage
A DNA sequence corresponding to a part of a or plasmid vector containing a cloned genomic
specific gene can be obtained by reverse or cDNA sequence is treated with a small amount
transcription of mRNA. cDNA thus obtained can of pancreatic DNase which hydrolyzes the
be cloned and used as a probe. phosphodiester bonds between nucleotides. At
Synthetic oligonucleotides as probes very low concentration the DNase produces only
scattered “nicks” in one or other strand of the
DNA probes with known nucleotide sequence can
duplex DNA. DNA polymerase and radioactively
also be synthesized chemically using automated
labeled deoxynucleotides are also added to the
DNA synthesizers. These synthetic probes will
DNA sample. Using the unharmed strand as
be efficient only when they are not more than 20-
template, the DNA polymerase synthesizes a new
40 nucleotides in length.
second strand using exposed 3' end at a nick
Labeling of probes site as primer, which then displaces the existing
The detection of homologous sequences after DNA from the 5’ end of the nick. Radioactive
hybridization with the probe is like finding a needle nucleotides are incorporated into the new strand,
in the hay-stock. Therefore, for the success of so, a single standard probe is created when the
DNA probe assay it is necessary to develop duplex DNA is denatured.
simple, safe and sensitive techniques for their
use. As probes transmit no signal of their own Choice of Label
they have to be either labeled with radioactive Probes can be labeled either by radioactive
isotopes or coupling of non-radioactive signal isotopes or can also be labeled with non-
molecules to the probes without impairing the radioactive molecules such as biotin, digoxegenin
hybridization ability of these probes. These signal etc.
molecules may include fluorescent antibodies, Radiolabeled Probes
enzymes that produce color changes in dyes and
The most sensitive detection method employed
chemiluminescent catalysts.
is radioactive label. Traditionally radioactively
Methods for Labeling of Probes labeled probes are used for a variety of
There are two methods for labeling of probes. experiments. The autoradiographic detection of
i.e. end labeling and nick translation. labeled probes depends on the isotope used and

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

the specific activity which has to be high enough Table 2: Merits of Radiolabeled and
to permit detection after hybridization within a Non-radiolabeled Probes
reasonable exposure of time or with a good Label Resolution Sensitivity Exposure Stability
signal. Several isotopes are available for
32 p + ++ 7 days 0.5 weeks
radioactive labeling. The use of 32P allows rapid
detection of signal yet cellular localization is 35 S ++ +++ 10 days 6 weeks

suboptimal because of the long path of the [32P] 3H +++ +++ 14 days >30 weeks
 -rays. Autoradiographs of high quality and
Biotin +++ ++ 0.16 days >52 weeks
improved cellular localization employ 35S labeled
Digoxigenin +++ ++ 0.16 days >52 weeks
probes. 35S emits  -rays of much shorter path
length. One of the major disadvantages of 35 S is
and 2.
non-specific binding of label to cell or tissues. It
has been suggested that pre-hybridization in the Non-Radiolabeled Probes
presence of non-labeled thio alpha UTP at pH 5.5 a) Biotin Labeled Probes
reduces this non-specific binding. It is also
Recent advances in nucleic acid technology now
possible to label nucleic acids with tritium or by
offer alternatives to radio activity labeled probes.
iodonization with [125 I] iodine. Tritium labeled
One of such procedure that is becoming popular
probes give intracellular localization, but for low
abundance target molecules may require is biotin labeling of nucleic acids. This system
exposure of up to 100 days. This may result in exploits the affinity which the glycoprotein avidin
high background and is unacceptably long for has for biotin. Avidin is commonly found in egg
most purposes. Disadvantage with radiolabel is white. Biotinylated probes are prepared through
Instability of label. Different labels along with the a nick-translation reaction by replacing
merits and demerits were presented in Tables 1 nucleotides with biotinylated derivatives. After
hybridization and washing, detection of hybrids
Table 1: Different Types of Labels
for Labeling of Probes is done by a series of cytochemical reactions
Radiolabeled Probes Non-radioactive Labelled Probes
which finally give a blue color whose intensity is
proportional to the amount of biotin in the hybrid.
3H, 32P, 35S,14C,1251 Digoxigenin

4-(Phenoxymethyl)piperidine Biotine
Advantages
These assays employ non-toxic materials, whose
5-Bromo deoxyuridine
half-life is longer.
Dinitro phenyl
• These probes can be prepared in advance in
Alkaline Phosphate
bulk and stored at -20°C for repeated uses.
Ethidium
• Detection of hybrids is much faster than by
Sulphonation
radioactive probes.
Luciferase

Sodium metabisulphate LIMITATIONS

Mercury Meta Acetate • A limitation of this technology is that very small
probe contains only a small number of
TetramethylRhodamine

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

biotinylated sites limiting the intensity of signal been devised for attaching other ligands (eg:
obtained. It has been solved by adding long hapten determinants, 2, 4 dinitrophenol,
‘tails’ of biotinylated nucleotides to the probes arsenative derivatives etc) to nucleotides without
through enzymatic methods. Sometimes the hampering their ability to be incorporated into
probe does not need to be labeled with biotin DNA. These alternatives require binding of
but only coupled with a tail. attached ligands to specific proteins that can be
tagged with enzymes or fluorescent molecules.
• Another disadvantage of biotin labeled probes
It is possible to monitor many probes
is that cytochemical visualization reactions
simultaneously by using several different ligands
lead to precipitation of insoluble material which
since each ligand would yield a different signal. A
cannot be removed and therefore the filter
chemiluminscent probe system has also been
cannot be reused, whereas with radio labeled
developed in which two different probes
probes, the filters can be used repeatedly for
complementary to a continuous segment of DNA
hybridization with a number of probes one at
hybridize to adjacent segments of a gene. The
a time.
first label is a chemiluminescent complex that
b) Digoxigenin Labeled Probes emits light at a specific wavelength this emission
Digoxigenin is another chemical derived from excites the label molecule on the second probe
plants and used for non-radioactive labeling of to emit light at a different wave length which can
probes. An antibody associated with an enzyme be detected using a photomultiplier device: This
(antidigoxigenin - alkaline phosphatase conjugate) process called non-radioactive energy transfer
is used for the detection of the presence of can occur only if the two probes hybridize
digoxigenin. The probes may be labeled with correctly and the two labels are close to each
digoxigenin - II - dUTP supplied with a digoxigenin other. This system has great fidelity and provides
kit (these kits are available from any commercial basic technology for a homogeneous assay. In
firm, eg. BoehringerMannheem). The labeled and addition DNA does not need to be immobilized
denatured probe may be used for hybridization. and no washing steps are necessary which may
After hybridization the membrane or the slide may be an additional advantage for large scale testing.
be transferred into detection buffer containing
20 ug/ ml of antidigoxigenin fluorescein and 5% Applications of Molecular Probes
(W/V) BSA (bonine serum albumin). This is Molecular probes are used in restriction fragment
incubated for 1 h at 37°C and then the membrane length polymorphisms (RFLPs) and related
or slide is washed in detection buffer three times Analysis
(8 min each at 37°C) and alkaline phosphatase
a) RFLPs for Evolutionary Studies
activity was detected using 0.17 mg/ml BCIP (5-
The restriction fragment length polymorphisms
bromo 4-chloro 3-indoyl phosphate) and 0.33 mg/
(RFLPs) can be studied in a set of related
ml NBL (nitro blue tetrazolium) as dye substrate.
species using a random or a specific DNA probe.
c) Alternatives to Biotin and Digoxigenin The similarities and differences can be used to
Labeling
infer phylogenetic relationships. This has actually
The techniques of non-radio isotopic labeling have been done in a number of cases both in plants
been further expanded and new methods have

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

and in animals(Gill et al., 1991; Fukuchi et al., identified through RFLP mapping analysis which
1993; Moore et al., 1991; Mason-Gamer demonstrates power of RFLP/Linkage analysis.
et al.,1998; Deshpande et al., 1998; Drinkwater
Use of Molecular Probes in Molecular
et al., 1991; Mburu and Hanotte, 2005; Guimaraes,
Cytogenetics
2007)
a) Isolation of Genes Using DNA or RNA
b) RFLP Maps and Linkage of RFLPs with Probes
Specific Genes Specific molecular probes can be used for
RFLPs have been used to prepare chromosome isolation of specific genes. These probes may
maps in humans, mice, fruit fly and in plants be available either from same species or from
including maize, tomato, lettuce, and rice. The another species can be used for isolation of
use of Mendelian markers for genetic mapping is genes. If probes obtained from one species used
sometimes limited due to non-availability of for isolation of gene from the same species they
mutants. The list of markers can be increased or are called as homologous probes. If probes
extended by using molecular markers which are obtained from another species used for isolation
examined in the form of RFLPs. Once a large of genes in other species they are defined as
number of RFLPs are available in a species the heterologous probes.
parents, F1, F2 generations can be used to study
These heterologous probes have been found
their inheritance and linkage relationship and
to be effective in identifying gene clones during
genetic linkage maps can be prepared. In this way
colony hybridization or plaque hybridization or on
they can be used for plant and animal breeding
southern blots. For instance, the gene for
(Mburu and Hanotte et al., 2005; Guimaraes et
chalcone-synthase (CHS) has been isolated from
al., 2007; Parasnis et al., 1999; Sen et al., 1997;
Antirrhinum majus and Petunia hybrida using
Pujar et al., 1999; Sant et al., 1999)
heterologous cDNA probes from parsley similarly
c) RFLP Markers to Map the Genes in heterologous Antirrhinum cDNA probe was used
Diseased Persons and Identification of
for isolation of CHS gene from barley and
Disease
heterologous probes from maize were used for
DNA polymorphisms (many forms) are
isolation of barley genes Wx (Waxy Genes) and
differences in DNA sequence that result from
Al (aleurone gene). Heterologous probes should
point mutations, random deletions or insertions
ordinarily be used with cDNA library and not with
or the presence of varying number of repeated
the genomic library since in the latter case
copies of a DNA fragments (tandem repeats). A
unrelated genes or pseudogenes (which do not
polymorphism in the coding region of a gene may
express) may be isolated and cloned. These
be detected as an alteration in the amino acid heterologous clones if available in expression
sequence of the encoded protein. It is now vector [Eg. PGEM Blue] can also be used for
possible to detect polymorphisms in unexpressed getting RNA probes which have been bound to
regions of DNA by this analysis. Genetic be more sensitive and efficient.
disorders like sickle cell anemia, Thalassemia’s,
b) In situ Hybridization
Huntington’s disease and cystic fibrosis were

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

In situ hybridization (ISH) is a technique which As represented in Table 3 the DNA probes are
permits detection of DNA or RNA sequences in being extensively utilized for diagnosis of
cell smears, tissue sections and metaphase diseases caused by parasitic protozoa and
chromosome spreads. The method is based on helminthes. They are also used for antenatal
the formation of double stranded hybrid molecules diagnosis of congenital diseases to allow advice
which form between a DNA or RNA target on abortion of fetus, if desired. Similarly probes
sequence and the complementary single have been designed for the diagnosis of number
standard labeled probe. In a number of cases rye of sexually transmitted diseases.
chromosomes in wheat background have been
Readymade DNA probes for herpes virus and
identified using this technique.
other human, animal plant viruses are available.
Satellite chromosomes with NORs (nuclear Probes are also available for a number of human
organizing regions) are recognized using probes parasites from the groups protozoa and
for ribosomal DNA in wheat, barley etc. Telomeres helminthes. In India, a diagnostic probe for the
have been identified in human and other detection of malaria has been constructed at the
eukaryotes using a telomeric sequence as a Astra Research Centre India (ARCI).
molecular probe showing that telomeres of all
chromosomes carry the same sequence. In USE OF MOLECULAR PROBES
wheat and related species chromosomes of D IN DNA FINGER PRINTING
genome can be identified using a D genome DNA fingerprinting is a way of identifying a specific
specific probe (PASI separated from Aegilops individual, rather than simply identifying a species
squarrosa).
or some particular trait. It is also known as genetic
This method is particularly useful if target fingerprinting or DNA profiling. Inventor of this
sequences are distributed in a non-random way technology is Sir Alec Jeffreys in 1985. DNA
in tissues for the visualization of heterogenicity fingerprinting is currently used both for identifying
and the study of cell differentiation. The method paternity or maternity and for identifying criminals
of ISH for RNA-RNA hybridization is generally or victims.
applicable to rapid screening of small numbers
The vast majority of a human’s DNA will match
of cultured cells for expression of oncogenic
exactly that of any other human, making
mRNA. It circumvents the problem of extracting
distinguishing between two people rather difficult.
rare RNAs in sufficient amounts for detection.
DNA fingerprinting uses a specific type of DNA
Similar methods have been used to visualize
sequence known as Variable Number Tandem
homeotic gene expression in developing larvae.
Repeats (VNTRs) can contain anywhere from
DNA-DNA ISH is also suitable for the detection of
twenty to one hundred base pairs. Every human
viral genomes in sections of routinely processed
being has some VNTRs. To determine if a person
archival paraffin blocks of human tissues.
has a particular VNTR, a southern Blot is
USE OF MOLECULAR PROBES performed and then the southern blot is probed
FOR HUMAN HEALTH CARE through a hybridization reaction with a radioactive

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

Table 3: DNA Probes for Different Diagnostic Tests

Parasite Probe Used HybridizationTechnique Used

I Protozoa

1. Leishmania Kinetoplast DNA (KDNA, a repetitive DNA Dot blots, southern blots In situ hybridization
(Kala-azar) sequence)

2. Trypanosoma Kinetoplast DNA (KDNA) a repetitive DNA Southern Blot (Restriction Fragment
(Sleeping sickness) and total parasite DNA Length Polymorphism
RFLP

3. Plasmodium Repetitive DNA, Synthetic oligonucleotides Dot blots, blood-samples lysed directly on
(Malarian Parasite; nitrocellulose filters
P. falciparum
P. vivax and P.
cynomorge)

II Helminths

1. Schistosomes Ribosomal RNA gene Dot blots, Southern blots

(human blood
flukes)

2. Nematodes: Crude DNA Blood Samples dot blots

Repetitive DNA sequences; Synthetic
Wuchereria and oligonucleotids; Ribosomal DNA;
Brugia (filaria)
onchocerca
(river blindness);
Trichinella

3. Taeniid Cestodes Ribosomal DNA Southern blots

(Taenia solium)

version of the VNTR in question. The pattern of high molecular weight DNA, complete digestion
which results from this process is what is often of the samples with appropriate enzymes and
referred to as a DNA fingerprint. perfect transfer and hybridization of the blot to
obtain distinct band with appropriate control. If
DNA fingerprinting has a high success rate and
necessary the analysis should be repeated. The
a very low false-positive rate making it an
DNA fingerprinting test is performed properly is
extremely popular form of paternity and maternity
infallible. However, there is need to interpret DNA
verification. In forensics DNA fingerprinting is very
typing with a thorough understanding of the
attractive because it requires only a piece of a
population involved.
person’s body like blood, hair, saliva, semen, body
tissue cells. DNA finger printing can be used for:

DNA finger printing is technically demanding. 1) Pedigree analysis and establishing paternity.
There are cases on record in the US where 2) Immigration authorities.
mistakes seem to have been prompted by human
3) In rape cases.
error. There is a need for quality control. Every
precaution should be taken to ensure preparation 4) Identification of mutilated dead bodies.

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Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

5) Social security record identification. 2. Belak S, Thoren P, Leblanc N and Viljoen G

(2009), “Advances in Viral Disease
6) In murder cases.
Diagnostic And Molecular Epidemiological
7) In characterization of cell cultures. Techniques”, Expert Rev. Mol. Diagn., Vol.
8) In animal breeding programmes. 9, No. 4, pp. 367-381.

9) In plant breeding programmes. 3. Bexfield N and Kellam P (2011),

“Metagenomics and the Molecular
10) In demographic studies.
Identification of Novel Viruses”, Vet. J., Vol.
11) Twin zygosity determination. 190, pp. 191-198.
12) Tissue culture cell line identification. 4. Chuba P J, Pelz K, Krekeler G, de Isele T S
13) Mopping fine grained sequence variation in and Gobel U (1998), “Synthetic
mini satellites. Oligodeoxynucleotide Probes For The
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their use has become today‘s most sophisticated Manners J M and Henry R J (2001),
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P, Gupta V S and Ranjekar P K (1998),
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“Evolutionary and Polymorphic Organization
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of the Knotted-1 homeobox in Cereals”,
modularity, minimal size, deliverability (access to
Theor Appl Genet., Vol. 97, pp. 135-140.
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