DNA probes are short segments of DNA or RNA that are labeled to allow for detection when bound to complementary nucleic acid sequences. Probes can be labeled through various methods, including fluorescent dyes, isotopic labeling using radioactive atoms, or non-isotopic labeling using molecules like biotin. Labeled probes are used in techniques like Southern blotting, PCR, and in situ hybridization to detect specific DNA or RNA sequences and analyze genetic material.

1. DNA probes
Presentedby
FathimaHameed

2. Outlines
 Introduction
 Method of nucleic acid hybridization
 Uses of DNA probes
 Features of DNA probes
 Labeling of DNA
 Methods of labeling DNA
 preparation of labeled nucleotides
 Uses of end labeled DNA
 Detection of probes

3. introduction
probes are short section of DNA or RNA with an additional
tagged or labeled chemically entity that are used to bind it’s
Complimentary strand and there by allows detection of candidate
Nucleic acid molecules.

4. The chemically synthesized entity are;
 fluorescent molecule
 an attachment to a colored bead
 quantum dots
 photo chromic compounds
 isotopic labeling
 non- isotopic labeling
It allows us to visualize when a probe attaches to DNA, RNA or other
target nucleic acids.

5. For hybridization, it involves interaction of single strands of two sources of
nucleic acids;
• Chemically synthesized
nucleic acids or modified
oligonucleotides.
• To identify similar or identical
complementary sequences.
probe
• Template nucleic acid
molecules.
• Form complex and
heterogeneous mixture.target

6. Gene probe:
 it generally longer than 500 bases.
 it consist most of a target gene.
Hybridization probes:
 probe is labeled in “standard hybridization assay”
 target is labeled in “reverse hybridization assays”
 nucleic acid probes can be single or double standard.
 working probe must be single stranded.

7. Method of nucleic acid
hybridization

8. Southern blotting
Northern blotting
Colony hybridization
Plaque hybridization
In situ hybridization
Sequencing by
hybridization
Uses of
probes:

9. features Hybridization probe
type DNA
origin cell based DNA cloning or PCR
characteristics of starting material Normally double stranded, 0.1 kb to
hundreds of kb for conventional DNA
clones, 0.1 kb to > 20 kb for PCR
products
labeling Usually by DNA polymerase based
DNA strand synthesis

10. Labeling of DNA
 Probes can be labeled at specific location within the oligonucleotides
 Some probes are of defined length.
 Some probes are heterogeneous population of labeled molecules.
two ways of labeling;
In vivo labeling In vitro labeling

11. In vivo labeling:
 DNA and RNA can be directly labeled inside tissue
culture cells by adding labeled deoxynucleotides in
culture plate in vivo.
 This method is restricted only to prepare labeled viral
DNA from virus infected cells and to study RNA
Processing events.

12. In vitro labeling:
 It is a more versatile.
 It involving in vitro labeling of purified RNA,DNA or
Oligonucleotide using DNA polymerase for
incorporation of labeled nucleotides.

13. In vitro labeling of DNA can be done by
various methods as follows,
Nick - translation
Random primed labeling
PCR mediated labeling

14. 1.DNA nick translation
 It involves insertion of random single strand breaks called “nicks”.
 The nicks in one strands of double stranded target DNA which exposes 3’-
OH termini and 5’-PO4 termini.
 The nicks are introduced by endonuclease like pancreatic
deoxyribonuclease I (DNase I).
enzymes are used for nick translation,
• Dnase I
• E.coli DNA polymerase I

15. Dnase I – exonuclease
DNA polymerase- multi subunit enzyme
 The exonuclease attacks the 5’
termini of a nick & removes the
nucleotides in 5’→3’ direction.
 DNA polymerase adds the
nucleotides to the free 3’- OH
group, in 5’→3’ direction.

16. Advantages:
 This method requires 100-fold less radioactive precursor.
 The amount of radio labeled incorporated depends on number of nicks
created by Dnase I.
Disadvantages:
 Only one complete regeneration is takes place.
 Reaction does not proceed further.

17. 2.Random primed DNA labeling
 This method is known as “oligo-labeling”
 It is based upon hybridization 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.

18. Random primed DNA labeling

19. Advantages:
 It produce labeled DNA’s of high specific activity.
 Primer represents all possible sequence combination & uniform labeling of
DNA occurs.
 Random priming is inherently simpler than nick translation, because the
requirements for two nuclease activity are eliminated.
 The probes generated by random priming method are more homogenous in
size.
 Probes are more reproducibly in hybridization reactions.

20. Disadvantages:
 Since binding of primer to template DNA is only random.
 The length of random primers is crucial, primers shorter than 6 bases are
very poor primers.

21. 3.PCR mediated DNA labeling
 The standard PCR reaction can be modified to incorporate labeled
nucleotides.
 This method commonly using in 2 ways,
Standard PCR based
DNA labeling
Primer mediated 5’
end labeling

22. Standard PCR based DNA labeling

23. Standard PCR based DNA labeling:
 The probe generation reaction is modified to incorporate one or more
labeled nucleotide precursors at a concentration same as oligonucleotide
concentrations.
Primer mediated 5’ end labeling:
 Radio labeled probes can be generated for both strands using equal
concentration of primers or heavily in favor of one strand of DNA using
higher concentration of one primer.
 It uses a 5’ end labeled primers.

24. Advantages:
 Defined segment of target DNA can be amplified independently of
restriction sites.
 Amount of template DNA is required very small.
 No need to isolate fragments of DNA or to sub-clone into vectors
containing bacteriophage promoters.

25. Preparation of labeled nucleotides
Isotopic labeling
Non-isotopic
labeling

26. Isotopic labeling:
 They can be detected directly in solutions or on x-ray film using
autoradiography.
 The strength of autoradiography signals depends on intensity of radiation
emitted by radioisotope and duration of exposure.
 Mostly using isotopes are 32P, 33P,35S& 3H.
Radioisotopes Half-life Energy of
emission
3H 12.4 years 0.019 MeV
32P 14.3 years 1.710 MeV
33P 25.5 years 0.248 MeV
35S 87.4 years 0.167 MeV

27. 32P
• Emits high energy ẞ- particles.
• High detection sensitivity.
• Used in southern blot& dot-blot hybridization, colony
hybridization.
35S
• Emits less energetic ẞ-radiation.
• Used in DNA sequencing & in-situ hybridization.
3H
• Low energy ẞ-particle emission.
• Long exposure time.

28. Non-isotopic labeling:
In this systems involved the use of non radioactive probes.
Two types of non radioactive labeling are conducted,
Direct non-isotopic labeling
Indirect non-isotopic labeling

29. Direct non-isotopic labeling:
 Where a nucleotide containing
label such as,
 Fluorescein
 Texas red
 Rhodamine
 These will be detected when
incorporated with the help of
spacer molecule.
 These modified nucleotides
having tag & fluoresce when
excited by light of certain
wavelength.

30. Indirect non-isotopic labeling:
 It involves chemical linkage of reporter molecule to a nucleotide.
 When this modified nucleotide is incorporated into DNA, then it is
specifically bound to a protein or other ligand.
 It has high affinity against the reporter group.
 Long spacer is introduced between nucleotide and reporter .

31. Indirect non-isotopic labeling

32. Two widely used non-isotopic labeling methods are,
• Biotin works as the reporter & streptavidin
is a bacterial protein is used for affinity
molecules.
• Enzymatic methods are mostly used to
label DNA probes with biotinylated
nucleotides.
Biotin- streptavidin
method
• A plant steroid obtained from digitalis
plant and is used as a reporter & an affinity
molecules.
Digoxigenin method

33. End labeled DNA can be used as;
 Molecular weight standards in southern blotting.
 Probes in gel retardation experiments.
 Traces for small quantities of DNA’s on gels.
 Probes for screening bacterial colonies or plaques.
 Substrates for Maxam-Gilbert sequencing.
 Probes for RNA mapping with S1 nuclease or mung bean nuclease.
 primers in primer –extension reactions.

34. Detection of non-radioactively labeled probes
after hybridization
 Affinity molecules are conjugated with a variety of marker groups or
molecules.
 They include various fluorophores or enzymes such as alkaline phosphatase
and peroxidase which can permit detection via,
 colorimetric assays
 fluorescent assays
 chemiluminescence

35. Thank you