Nucleic acid probes

1. • “For diagnostic tests, the agent that is used to
detect the presence of a molecule in the
sample”.
• “A DNA sequence that is used to detect the
presence of a complementary sequence by
hybridization with a nucleic acid acid sample
sample”.

3. • 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.

4. • 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 . For statistical uniqueness,
a minimum of 20 nucleotide bases are usually needed
for a probe.

6. • 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.
• short probes do have some disadvantages.
• They are subject to more nonspecific hybridizations, are limited
in specificity, and are more difficult to label.
• Long probes hybridize more stably than short probes at high
temperatures and low salt concentrations(low stringency).

7. – A nucleic acid probe
• Is a short, single-stranded molecule of
radioactively labeled or fluorescently labeled
DNA or RNA
Radioactive
probe (DNA)
Single-stranded
DNA
Mix with single-
stranded DNA from
various bacterial
(or phage) clones
Base pairing
indicates the
gene of interest
A T C C G A
A T G C G C T T A T C G
A
G
C C
T T A
T G
C A
T
A T C C G
A
A G G T A G G C T A
A

8. • The base sequence of the probe and the conditions
under which the probe is used determine its
specificity.

9. • Whatever label is used, it has to be attached to, or
incorporated into, the nucleic acid probe.Here are 5
methods of labelling probes
• Nick translation
• Primer extension
• RNA polymerase
• End labelling
• Direct labelling

10. • It involves introducing single-strand breaks
(nicks by endonuclease) in the DNA, leaving
exposed 3′ hydroxyl termini and 5′ phosphate
termini.
• nick serves as a start point for introducing new
nucleotides at the 3′ hydroxyl side using DNA
polymerase
• As a result, the nick will be moved progressively
along the DNA (‘translated’) in the 5′ → 3′
direction .
• the synthesis reaction allows the incorporation of
labeled nucleotides in place of the previously
existing unlabeled

12. • Based on hybridization of a mixture of all possible
hexanucleotides .
• the starting DNA is denatured and then cooled
slowly so that the individual hexanucleotides can
bind to suitably complementary sequences within
the DNA strands.
• DNA synthesis occurs in the presence of the four
dNTPs, at least one of which has a labeled group.

14. • DNA of interest is denatured to give single-strands.
• Random primer sequences are added (or sequences unique to a
particular sequence of interest).
• DNA polymerase is added together with labelled nucleotides.
• Complementary DNA strands are synthesized starting from the
primer sequences and incorporating the labelled nucleotides. There
is partial filling in of the gaps between the primers.
• DNA is then denatured to release the labelled probe molecules.

16. • Single-stranded oligonucleotides are usually end-labeled
using polynucleotide kinase (kinase end-labeling).
• label is provided in the form of a 32
P at the γ-phosphate
position of ATP and the polynucleotide kinase catalyses an
exchange reaction with the 5′-terminal phosphates.
• The same procedure can also be used for labeling double-
stranded DNA.
• Here fragments carrying label at one end only can then be
generated by cleavage at an internal restriction site,
generating two differently sized fragments which can be
separated by gel electrophoresis and purified.

18. • The preparation of labeled RNA probes (riboprobes) is
most easily achieved by in vitro transcription of insert
DNA cloned in a suitable plasmid vector.
• The vector is designed so that adjacent to the multiple
cloning site is a phage promoter sequence, which can be
recognized by the corresponding phage RNA polymerase.
• By using a mix of NTPs, high specific activity
radiolabeled transcripts can be generated.
• Labeled sense and antisense riboprobes can be generated
from any gene cloned in such vectors and are widely used
in tissue in situ hybridization
• DNA template + RNA polymerase + labelled
ribonucleotides ======= labelled RNA probe

20. • Radiolabels
• Non-radioactive labelsNon-radioactive labels
• ChemiluminescenceChemiluminescence
• Fluorescence
• Antibodies

21. • Probe nucleic acid can be labelled using radioactive
isotopes, e.g. 32
P, 35
S, 125
I, 3
H.
• Detection is by autoradiography or Geiger-Muller counters.
• Radiolabelled probes used to be the most common type but are less
popular today because of safety considerations.
• However, radiolabelled probes are the most sensitive, e.g.32
P labelled
probes can detect single-copy genes in only 0.5 mg of DNA.
• High sensitivity means that low concentrations of probe-target
hybrid can be detected.

22. • These are harmless than radiolabels and do not require
dedicated rooms, glassware and equipment or staff
monitoring, etc. but they are not generally as sensitive.
• Some examples:
• Biotin This label can be detected using avidin or
streptavidin which have high affinities for biotin.
• Enzymes The enzyme is attached to the probe and its
presence usually detected by reaction with a substrate
that changes colour. Used in this way the enzyme is
sometimes referred to as a "reporter group".
• Examples of enzymes used include alkaline
phosphatase and horseradish peroxidase.

23. • In this method chemiluminescent chemicals attached
to the probe are detected by their light emission using
a luminometer.
• luminometer.

24. • Fluorescence Chemicals attached to probe fluoresce under
UV light. This type of label is especially useful for the direct
examination of microbiological or cytological specimens
under the microscope – a technique known as fluorescent in
situ hybridization (FISH).

25. • Antibodies An antigenic group is coupled to the
probe and its presence detected using specific
antibodies. Also, monoclonal antibodies have been
developed that will recognize DNA-RNA hybrids.

26. • Probe and target sequence hybridize with each
other but how this is brought about can vary.
There are 4 main formats:

27. • Target (usually) is bound to a solid support such
as a microtitre tray or filter membrane.
• Probe is added in solution and binds to target (if
present) on solid support.
• After washing to remove unbound probe,
hybridization is detected on the solid support
using whatever method is appropriate for the
probe label.

28. • Both the probe and the target are in solution.
Because both are free to move, the chances of
reaction are maximized and, therefore, this
format is generally faster than others.

29. • In this format probe solution is added to fixed tissues,
sections or smears which are then usually examined
under the microscope.
• The probe label, e.g. a fluorescent marker, produces a
visible change in the specimen if the target sequence is
present and hybridization has occurred.
• However, the sensitivity may be low if the amount of
target nucleic acid present in the specimen is low. This
can be used for the gene mapping of chromosomes, and
for the detection of microorganisms in specimens.

30. • After size fractionation of nucleic acids by
electrophoresis, they are transferred to a filter
membrane which is then probed.
• The presence of target is confirmed by
detection of probe on the filter membrane, e.g.
radiolabelled probe can be detected by
autoradiography and the location of the target
sequence in the bands in the original gel
determined.

31. USES OF PROBES
1. Southern blots
Detection of gel-fractionated DNA molecules transferred to a
membrane. This includes restriction fragment length
polymorphism (RFLP) analysis.
2. Northern blots
As above but used for RNA.
3. Dot blots
Detection of unfractionated nucleic acid immobilized on a
membrane.
4. Colony and plaque blots
Detection of immobilized nucleic acid on a membrane that has
been released from lysed bacteria or phages.
5. In situ hybridization
Direct detection of nucleic acid in clinical specimens.

32. 1. Detection of specific nucleic acid sequences
Probes can be especially useful for detecting
microorganisms that grow slowly (e.g.
Mycobacterium tuberculosis) or which cannot
be cultured on artificial growth media (e.g. all
viruses).

33. • A change to the DNA sequence is a mutation, e.g. deletion,
insertion, substitution. Changes in certain gene sequences can
cause inherited diseases and their detection by probes can be
diagnostic.
• cystic fibrosis (due to a 3 bp deletion).
• muscular dystrophies (due to various intragenic deletions).
• phenylketonuria (due to various mutations).
• apolipoprotein variants (some are due to a 1 bp mutation).
• sickle cell anaemia (due to a 1 bp mutation).
• a1-antitrypsin deficiency (due to approx. 50 different variants).

34. • Examples of the Applications of Nucleic Acid Probes in Medical Research
• Detection of tumor suppressor genes in human bladder tumors
• Identification of Leishmania parasites
• Detection of malignant plasma cells of patients with multiple myeloma
• Diagnosis of human papillomavirus
• Visual gene diagnosis of HBV and HCV
• Detection and identification of pathogenic Vibrio parahaemolyticus
• Detection of Vibrio cholerae
• Molecular analysis of tetracycline resistance in Salmonella enterica
• Identification of fimbrial adhesins in necrotoxigenic E. coli
• Epidemiological analysis of Campylobacter jejuni infections
• Molecular analysis of NSP4 gene from human rotavirus strains
• Physical mapping of human parasite Trypanosoma cruzi
• Detection and identification of African trypanosomes
• Detection and identification of pathogenic Candida spp.
• Identification of Mycobacterium spp.

35. • Tandem repeat sequences are 30-50 bps in
length. Their size and distribution are
distinctive for an individual.
• They can be detected using probes and PCR.
They are the basis of so-called "DNA
fingerprinting"

36. • which was developed by Alec Jeffreys at the
University of Leicester, UK
• It is used in forensic science to confirm the
identity of a suspect from specimens left at the
scene of a crime, e.g. any body fluid, skin,
hair.
• This technique can also be used for paternity
tests, sibling confirmation (or exclusion) and
tissue typing.

37. • Sambrook, J. and Russell, D. W. (2001) Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
• Marilena Aquino de Muro” Probe Design, Production,
and Applications”
• V.SINGH” What are DNA probes?”
• Wetmur JG”DNA probes: applications of the principles
of nucleic acid hybridization”.
• "dna probes." Definitions.net. STANDS4 LLC, 2012.
Web. 12 Sep. 2012.
http://www.definitions.net/definition/dna probes
• http://www.springer.com/978-1-58829-288-9.