Fluorophore Based Chemistries Used In Various Molecular Techniques

Fluorescent reporter molecules are used with various molecular technologies to monitor biological processes.

1. Fluorophore Based Chemistries Used In Various Molecular Techniques PRIYANKA GUPTA PhD Research Scholar Dept. Of Lab. Medicine , AIIMS, New Delhi 02-03-2016 1

2. Fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Principle of fluorescence given by “ Aleksander Jablonski ” named as Jablonski energy diagram. Introduction 02-03-2016 2

3. They are sometime used alone, part of an enzyme or covalently bonded with bioactive molecules. (e.g. a ntibodies, protein & nucleic acid) Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy. Fluorescent reporter molecules are also used with various molecular technologies to monitor biological processes for example : PCR (Polymerase Chain Reaction) 02-03-2016 3

4. Real time PCR Flow cytometry LAMP (Loop Mediated Isothermal Amplification ) Fluorescence In Situ Hybridization (FISH) DNA Microarray 02-03-2016 4

5. Acridine 362 462 Caumarine 432 472 Cy3 552 570 Cy5 646 667 EtBr 493 620 Fluorescein 495 520 FAM 494 525 TAMRA 565 580 ROX 587 607 Texas Red 583 603 TET 521 536 JOE 528 554 HEX 535 556 Light cycler 610 590 610 Light cycler 640 625 640 Oregon Green 500 521 Rhodamine 503 580 VIC 538 554 Alexa Fluor species varies varies Yakima yellow 530 549 Dye Absorption (nm) Emission (nm) Dye Absorption (nm) Emission (nm) Fluorophores 02-03-2016 5

6. 02-03-2016 6 Fluorophore Dyes used in Flowcytometry :

7. Mostly used f luorophore dyes in DNA microarray is Cyanine dyes e.g. Cy3, Cy5, etc. Alexa fluore dyes are generally used in Fluorescence In Situ Hybridization. In the real time PCR the fluorophore molecules are categorised into two types: Donor or Reporter molecule : Fluorescent molecule, emit energy as fluorescence. e.g. FAM, JOE, etc. Acceptor or Quencher molecule : The a cceptor may or may not fluorescent molecule. When it is non fluorescent it is quencher, the electronic energy of quencher is dissipated in the form of heat. e.g. BHQ-1, BHQ-2, Dabsyl , etc. 02-03-2016 7

8. Reporters and quenchers for real time PCR: 02-03-2016 8

9. Donor and Acceptor for real time PCR: 02-03-2016 9

10. Ethidium Bromide ( EtBr ) : Ethidium bromide is a heteroaromatic cationic dye. Ethidium bromide is the most commonly used dye for DNA and RNA detection in gels. Ethidium bromide intercalate between the base pairs of double helix DNA or RNA. 02-03-2016 10 Fluorophore dye for PCR

11. Ethidium bromide has UV absorbance at 493 nm and emission at 620 nm. Ethidium bromide is a powerful mutagen, extremely toxic by inhalation, ingestion and skin contact, and a suspected carcinogen and reproductive toxin. Agarose gel with UV illumination, stained by EtBr 02-03-2016 11

12. Types of Fluorescence based Chemistries DNA Intercalating dyes Fluorophore labeled oligonucleotides SYBER Green 1 SYTO BEBO BOXTO PNA Hyb Probe or FRET TaqMan Cyclicons Scorpion Molecular Beacon LNA LUX Probes Primer-probes NA analogues EvaGreen 02-03-2016 12

13. These are the intercalating dyes. Bind with the minor groove of dsDNA and leads to fluorescence emission. The most common examples of these dyes is SYBER Green 1. The use of these dyes allow the detection of specific products, nonspecific products and primer-dimer produced during the reaction. DNA Binding Dyes 02-03-2016 13

14. SYBR Green I It is asymmetrical cyanine dye with two positive charges. It absorbs blue light at 497 nm and emits green light at 520 nm. Bind with minor groove of DNA. This dye is useful to perform melting curve analysis. Melting curve analysis is used for discriminating two or more different DNA sequence in a single PCR reaction of multiplex assays. 02-03-2016 14

15. Applications: P athogen detection. G ene expression. SNP detection GMO detection. 02-03-2016 15

16. These are small fluorescent molecules attached to oligonucleotides to be used function as probe. Two types of fluorophores : Donor or reporter Acceptor or quencher. The transfer of excited state energy from a donor to acceptor use FRET (Fluorescence Resonance Energy Transfer) mechanism. Two different types FRET mechanism use: Fluorophore - labeled Oligonuleotide 02-03-2016 16

17. FRET : Transfer energy emitted as fluorescence because the acceptor molecule is fluorescent . FRET-quenching : Electronic energy of the quencher is dissipated as heat because quencher is a nonfluorescent molecule. 02-03-2016 17

18. Probes Holland et al. (1991) These probes are oligonucleotides containing a reporter fluores cent moiety at the 5’-end and an quencher at 3’ end. These are designed to bind to a specific region of the target DNA. In solution the fluorescent signal is quenched due to the FRET-quenching phenomenon. 1. TaqMan or Hydrolysis probes: 02-03-2016 18

19. In the extension phase , the bound hydrolysis probe is removed by the 5’-3’ exonuclease activity of DNA polymerase and reporter dye generate florescence. Applications : Use in singleplex & multiplex format for viral detection . Viral/ bacterial load quantitation . Genotyping. Gene Expression. Mutation detection. 02-03-2016 19

20. 2. Hyb Probe or FRET : Heller and Morrison. (1985) These probes consists a pair of oligonucleotides bind to adjacent target DNA sequences. The first probe carries a donor fluorophore at its 3’ end and the second probe contain acceptor fluorophore at its 5’ end . These probes hybridize to the target DNA sequences in a head-to tail combination bring the fluorophore into close proximity, producing fluorescence due to FRET mechanism in annealing phase. 02-03-2016 20

21. Applications : Use in singleplex & multiplex format for viral detection . Viral/ bacterial load quantitation . Microarray validation. Gene Expression. Mutation detection. Melting curve analysis. 02-03-2016 21

22. 3. Molecular Beacon probes : Tyagi and K ramer . (1996) They are single standard hairpin shaped oligonucleotide probes which contain : one loop, 18-30 bp complementary to the target DNA sequence a stem contain two complementary sequences at end of the probe . a reporter dye at 5’ end and quencher at 3’ end. 02-03-2016 22

23. During the annealing phase, the molecular beacon probe unfolds and binds to the target DNA sequence, leading to fluorescence emission. Applications : Use in singleplex & multiplex format for viral detection . Viral/ bacterial load quantitation . Microarray validation. Gene Expression. Mutation detection. Melting curve analysis. Allelic discrimination. 02-03-2016 23

24. Primer-probes Primer- probes are oligonucleotides that combine a primer probe in a single molecule. These are nonspecific, so melting curve analysis to determine the efficiency of the reaction is recommended. 02-03-2016 24

25. 1. Scorpion Primer-probes Whitcombe et al. (1999) The hairpin structure of scorpion : a reporter at 5’-end an internal quencher at the 3’-end 3’ end attached to the 5’-end of HEG ( hexa glycol) blocker. 02-03-2016 25

26. After probe binding to target, DNA polymerase amplify the target sequence. In the next denaturation, the specific sequence of the probe bind to the complementary region within same strand of newly amplified DNA, leading to fluorescence emission. 02-03-2016 26

27. It is an inexpensive system in which primer-probe combines and detection mechanism in the same molecule. Applications : Pathogen detection in singleplex & multiplex format. Viral/ bacterial load quantitation. Genotyping. Mutation detection. 02-03-2016 27

28. 2. LUX ( Light-Upon-Extension) Nazarenko et al. The 3’-end act as primer and contain a single reporter located in the guanosine rich region of the primary sequence. TM The hairpin structure confer the ability to decrease the fluorescence signal when the primer-probe is free. Maximum fluorescence emission takes place when it incorporate with dsDNA during extension phase. 02-03-2016 28

29. Applications : It can be used for pathogen detection in singleplex & multiplex format. Viral/ bacterial load quantitation. Genotyping. Mutation detection . Gene expression analysis. GMO detection. 02-03-2016 29

30. 3. Cyclicons Kandimalla & Agrawal . (2000) It contain a long primer-probe complementary to target sequence and a short modified oligo attached through 5’-5’ ends. Which binds to 3’-end of the primer-probe forming cyclic structure with two 3’-ends. Cyclicons have a reporter at free 3’ –end and a quencher at 5’-end. 02-03-2016 30 Quencher Reporter 5’ 5’ 3’ Cyclicon primer-probe FRET

31. It binds to the complimentary sequence the cyclic structure opened up and the fluorophores are separated far enough to disrupt FRET-quenching resulting fluorescence emission. Applications : It can be used for pathogen detection in singleplex & multiplex format. Viral/ bacterial load quantitation. Genotyping. Mutation detection, SNP detection. It can be directly fixed to solid supports o the chips for high-throughput screening in solid-phase PCR. 02-03-2016 31

32. Nucleic acid Analogues Nucleic acid analogues are compounds that are analogous to naturally occurring RNA and DNA. e.g. PNAs , LNAs and ZNAs, etc. 02-03-2016 32

33. PNAs: Ni elson et al. They are achiral and electrically neutral DNA analogues in which the sugar-phosphate backbone has been replaced by a peptide of N-(2-aminoethyl)- glycine units. PNAs able to interact with either dsDNA or RNA with higher affinity and greater specificity than conventional oligonucleotide . 02-03-2016 33

34. The mechanism of primer-probes or probes in which PNA molecules have been introduced is identical to the method of action of conventional probes. Applications: Induce DNA recombination or block PCR amplification of specific gene. Uniquely, allelic discrimination of single nucleotide polymorphism can be accomplished by using PNA molecular beacon Discrimination between DNA and cDNA sequence in prokaryotes. 02-03-2016 34

35. 2. LNA s: Wengel and co-workers in 1998. LNAs are DNA or RNA sequence in a conformation that contain one or more modified nucleotides. They have a methylene bridge between atoms 2’-O and 4’-C in the ribose ring to form a bicyclic ring. LNA containing primer-probes or probes exhibit the same mode of action as that of convention probes. The use of LNA Molecular beacon and LNA TaqMan probes has been reported for SNP detection, GMO detection and viral quantification. 02-03-2016 35

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