2. MOLECULAR PROBES
• Small DNA / RNA segments.
• Used to detect complementary sequences
in nucleic acid samples.
• Abs – probes to recognize specific protein
sequences.

3. • Both DNA & RNA used as probes.
• ssDNA probes – more convenient &
preferable.
• Denatured dsDNA also used.
• RNA probes ordinarily ss.

5. • Genomic DNA probes.
• cDNA probes.
• Synthetic oligonucleotides as probes.
• RNA probes or riboprobes.

6. 1.GENOMIC DNA PROBES
• Extract DNA.
• Digest with restriction enzyme
• Run AGE/PAGE.
• Isolate DNA
• Clone it in a vector.
• Multiplication in bacteria.

7. Chimeric vector obtained from bacteria
used in following ways :
• Directly used as probe.
• Cloned segment separated & used as
probe.
• Chimeric DNA amplified (PCR)-product
used as probe

8. 2. cDNA probes
• cDNA - synthesized from isolated mRNA
using reverse transcriptase.
• Cloned & used as probe.

10. 3. SYNTHETIC OLIGONUCLEOTIDES AS
PROBES
• Probes with known sequence synthesized
chemically.
• Using automated DNA synthesizers

11. 4. RNA PROBES / RIBOPROBES
• DNA template cloned in expression vector.
• Vector has diff.& specific prokaryotic
promoter beyond 2 ends of DNA insert.
• Recombinant vector is linearized &
transcribed with appropriate RNA pol. to
obtain RNA molecules complementary to
one or other strand of DNA insert.

14. LABELLING OF PROBES
• RADIOACTIVE LABELLING
• NON-RADIOACTIVE LABELLING

15. RADIOACTIVE LABELLING
• Commonly used radioisotope labels –
32
P,3H,35S,125I.
• 3 methods for labelling –
Nick translation
Oligonucleotide labelling
Riboprobe preparation

16. Nick translation
• Create nick in probe DNA.
• Extension of broken ends labelled
deoxyribonucleotides & DNA pol.I

17. Oligonucleotide labelling
• Short random oligonucleotides used as
primers for copying probe DNA in the
presence of labelled deoxyribonucleotides.

18. Riboprobe preparation
• Synthesis of labelled RNA,using DNA
probe as template,in presence of labelled
ribonucleotides.
• After hybridization with labelled
probe,hybrids are detected by
autoradiography

19. Disadvantages of radioactive
labelling
• Radioisotopes difficult to handle &
expensive to dispose off.
• If there are few counts in hybrid detection -
autoradiography takes long time.
• R.isotopes have short halflife & therefore
expts should be completed fast.

20. Non – radioactive labelling
• The most commonly used labels for the
generation of non-radioactively DNA or
RNA hybridization probes are fluorophores
and haptens,
• the latter meaning Biotin and Digoxigenin.

21. Fluorescent probes
Fluorescent probes are detected directly after incorporation
by fluorescence spectroscopy.
Classical dyes such as rhodamine, fluorescein and cyanine
derivatives have been the most widely used

23. Biotin labelled probes.
• Prepared through nick translation rexn –
nucleotides replaced with biotinylated
derivatives.
• Detection of hybrids done by series of
cytochemical rexns which gives final blue colour.
• Colour intensity proportional to amount of biotin
in hybrid.

25. Digoxygenin labelled probes
BCIP+NBT

28. HRP
• Probe DNA + HRP
• HRP + luminol → chemiluminiscence
• Signal recorded on photo. film

29. Chemiluminescent labelling

31. • Identification of recombinant clone carrying
desired DNA insert.
• Confirmation of integration of DNA insert into
host genome.
• Development of RFLP maps.
• DNA fingerprinting for
identification of plant varieties,
criminals,parental relationships etc.

32. • Insitu hybridization for determining the
locations of specific sequences in specific
chromosomes.
• Accurate diagnosis of diseases caused by
parasites,pathogens or defective viruses.
• Preparation of genome maps of
eukaryotes,including man.