MOLECULAR PROBES• Small DNA / RNA segments.• Used to detect complementary sequences in nucleic acid samples.• Abs – probes to recognize specific protein sequences.
• Both DNA & RNA used as probes.• ssDNA probes – more convenient & preferable.• Denatured dsDNA also used.• RNA probes ordinarily ss.
• Genomic DNA probes.• cDNA probes.• Synthetic oligonucleotides as probes.• RNA probes or riboprobes.
1.GENOMIC DNA PROBES• Extract DNA.• Digest with restriction enzyme• Run AGE/PAGE.• Isolate DNA• Clone it in a vector.• Multiplication in bacteria.
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
2. cDNA probes• cDNA - synthesized from isolated mRNA using reverse transcriptase.• Cloned & used as probe.
3. SYNTHETIC OLIGONUCLEOTIDES AS PROBES• Probes with known sequence synthesized chemically.• Using automated DNA synthesizers
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.
LABELLING OF PROBES• RADIOACTIVE LABELLING• NON-RADIOACTIVE LABELLING
RADIOACTIVE LABELLING• Commonly used radioisotope labels – 32 P,3H,35S,125I.• 3 methods for labelling – Nick translation Oligonucleotide labelling Riboprobe preparation
Nick translation• Create nick in probe DNA.• Extension of broken ends labelled deoxyribonucleotides & DNA pol.I
Oligonucleotide labelling• Short random oligonucleotides used as primers for copying probe DNA in the presence of labelled deoxyribonucleotides.
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
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.
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.
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
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.
• Identification of recombinant clone carrying desired DNA insert.• Confirmation of integration of DNA insert into host genome.• Development of RFLP maps.• DNA fingerprinting foridentification of plant varieties,criminals,parental relationships etc.
• 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.