DNA Probe By Jwala Jayadeep KUFOS

1. DNA PROBE
BY JWALA
1ST MSc. MARINE MICROBIOLOGY

2. INTRODUCTION
DEFINITION:-
• In molecular biology, a probe is a fragment of DNA or RNA
of variable length (usually 100-1000 bases long) which
can be radioactively labelled.

3. TYPES OF PROBES
• There are three types of nucleic acid probes:-
1. Oligonucleotide probes
2. DNA probes
3. RNA probes.

4. DNA PROBES
• DNA probes are stretches of single stranded DNA used to
detect the presence of complementary nucleic acid
sequences (target sequences) by hybridisation.
• These are longer than the oligonucleotide.
• DNA probes are usually labelled, for example with
radioisotopes, epitopes, biotin or fluorophores to enable
their detection.

5. The chemically synthesized entity are:-
• Fluorescent molecule
• An attachment to a coloured bead
• Quantum dots
• Photo chromic compounds
• Isotopic labelling
• Non- isotopic labelling
It allows us to visualize when a probe attaches to DNA,
RNA or other target nucleic acids.

6. THE DNA MOLECULE
• The basis of molecular biology is deoxyribonucleic acid or
DNA.
• The strands of DNA can be separated by heat (thermal
denaturation) or by raising the pH or lowering the ionic
strength of the DNA solution.
• The DNA molecule is most stable in its native double
stranded state. Thus when single stranded DNA is placed
in solution, under appropriate temperature and salt
conditions, the complementary strands will recombine to
form a duplex molecule.
• Complementary sequences of DNA can bind to RNA
counterparts.

7. The hybridisation reaction consists of four components:-
• The probe
• The target (which is contained in the sample)
• The reporter molecule and
• The hybridisation method.

10. DESIGNING A PROBE
• All organisms contain some unique sequences of DNA or
RNA within their genome that distinguish them from all
other organisms.
• Hybridisation is a process whereby two single strands of
nucleic acid come together to form a stable double-
stranded molecule. As long as the sequences of bases
along each stretch of nucleic acid are complementary,
they will bind and stay together.

11. LABELLING OF DNA
• Probes can be labelled at specific location within the
oligonucleotides.
• Some probes are of defined length.
• Some probes are heterogenous population of labeled
molecules.
• Two ways for labelling
1. In vivo labelling
2. In vitro labelling

12. IN VIVO LABELLING
• DNA can be directly labelled inside tissue culture cells by
adding labelled deoxynucleotides in culture plate in vivo.
• This method is restricted only to prepare labelled viral
DNA from virus infected cells and to study RNA
processing events.

13. IN VITRO LABELLING
• It involves the process of labelling of purified DNA using
DNA polymerase for incorporation of labelled nucleotides.
• This process can be done through various methods:-
1. Nick-translation
2. Random printed labelling
3. PCR mediated labelling

14. DNA nick translation
• It involves the insertion of random single strand breaks
called “nicks”
• The nicks in one strands of double stranded target DNA
exposes 3’ and 5’ ends.
• The nicks are introduced by endonuclease like pancreatic
deoxyribonucleaseI (Dnase I)

15. RANDOM PRIME DNA LABELLING
• This method is known as “oligo-labelling”
• It is based upon hybridisation of a mixture of all possible
hexanucleotides.
• The template DNA is initially denatured.
• The synthesis of new complementary DNA strands is
primed by bound hexanucleotides.
• Random hexanucleotides to bind at complementary
sequences at which extension takes place through PCR.
• It was catalyzed by klenow subunit of DNA polymerase I
*The klenow fragment is a large protein fragment produced
when DNA polymerase I from E.coli is ezymatically cleaved
by the protease subtilisn.

16. PCR MEDIATED DNA LABELLING
• The stranded PCR reaction can be modified to
incorporate labelled nucleotides.
• This method commonly uses 2 ways:
1. Standard PCR based DNA labelling
The probe generation reaction is modified to incorporate
one or more labelled nucleotide precursors at a
concentration same as oligonucleotide concentrations.

17. 2. Primer mediated 5’ end labelling
• Radio labelled probes can be generated for both strands
using equal concentration of primers or heavily in favour
of one strand of DNA using higher concentration of one
primer.
• It uses a 5’ end labelled primers.

18. PLASMIDS
• To produce unique sequences, cloning vectors are used.
• The commonly used vectors are Plasmids.
• Plasmids are covalently closed circular pieces of DNA that
replicate independently of the bacterial chromosome.
They are not required for cell replication.
• Size range:- 1000 bp to 40000 bp.
• Cloning vectors are small plasmids often just 2-5
kilobases that contain a selectable marker such as
ampicillin resistance and a stretch of DNA that can be
cleaved by many different restriction endonucleases.

21. DIAGNOSTIC APPLICATIONS OF DNA
PROBES
• Advantages:-
1. Rapid detection and identification of infectious agents
2. The ability to screen selected specimens using batteries
of probes
3. The detection of non viable or difficult to culture
organisms.

22. • Disadvantages:-
1. Use of isotopic detection methods for optimum
sensitivity
2. Limited diagnostic sensitivity of current assays
3. Slow turnaround time for some assay formats
4. Expense of current reagents
5. Limited availability of many probes
6. Lack of technical expertise in most diagnostic
laboratories.

23. THANK YOU