molecular probes kashmeera n.a
TRANSCRIPT
![Page 1: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/1.jpg)
![Page 2: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/2.jpg)
MOLECULAR PROBES
• Small DNA / RNA segments.
• Used to detect complementary sequences in nucleic acid samples.
• Abs – probes to recognize specific protein sequences.
![Page 3: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/3.jpg)
• Both DNA & RNA used as probes.
• ssDNA probes – more convenient & preferable.
• Denatured dsDNA also used.
• RNA probes ordinarily ss.
![Page 4: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/4.jpg)
![Page 5: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/5.jpg)
• Genomic DNA probes.
• cDNA probes.
• Synthetic oligonucleotides as probes.
• RNA probes or riboprobes.
![Page 6: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/6.jpg)
1.GENOMIC DNA PROBES
• Extract DNA.
• Digest with restriction enzyme
• Run AGE/PAGE.
• Isolate DNA
• Clone it in a vector.
• Multiplication in bacteria.
![Page 7: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/7.jpg)
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
![Page 8: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/8.jpg)
2. cDNA probes
• cDNA - synthesized from isolated mRNA using reverse transcriptase.
• Cloned & used as probe.
![Page 9: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/9.jpg)
![Page 10: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/10.jpg)
3. SYNTHETIC OLIGONUCLEOTIDES AS PROBES
• Probes with known sequence synthesized chemically.
• Using automated DNA synthesizers
![Page 11: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/11.jpg)
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.
![Page 12: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/12.jpg)
![Page 13: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/13.jpg)
![Page 14: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/14.jpg)
LABELLING OF PROBES
• RADIOACTIVE LABELLING
• NON-RADIOACTIVE LABELLING
![Page 15: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/15.jpg)
RADIOACTIVE LABELLING
• Commonly used radioisotope labels – 32P,3H,35S,125I.
• 3 methods for labelling –
Nick translation
Oligonucleotide labelling
Riboprobe preparation
![Page 16: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/16.jpg)
Nick translation
• Create nick in probe DNA.
• Extension of broken ends labelled deoxyribonucleotides & DNA pol.I
![Page 17: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/17.jpg)
Oligonucleotide labelling
• Short random oligonucleotides used as primers for copying probe DNA in the presence of labelled deoxyribonucleotides.
![Page 18: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/18.jpg)
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
![Page 19: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/19.jpg)
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.
![Page 20: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/20.jpg)
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.
![Page 21: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/21.jpg)
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
Fluorescent probes
![Page 22: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/22.jpg)
![Page 23: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/23.jpg)
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.
![Page 24: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/24.jpg)
![Page 25: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/25.jpg)
Digoxygenin labelled probes
BCIP+NBT
![Page 26: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/26.jpg)
![Page 27: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/27.jpg)
![Page 28: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/28.jpg)
HRP
• Probe DNA + HRP• HRP + luminol → chemiluminiscence• Signal recorded on photo. film
![Page 29: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/29.jpg)
Chemiluminescent labelling
![Page 30: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/30.jpg)
![Page 31: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/31.jpg)
• 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.
![Page 32: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/32.jpg)
• 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.
![Page 33: Molecular probes kashmeera n.a](https://reader035.vdocument.in/reader035/viewer/2022081505/55504528b4c90580748b4d4a/html5/thumbnails/33.jpg)