Fundamental in Real Time PCR

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1 Fundamental in Real Time PCR

2 Real Time PCR Detection of PCR products via generation of fluorescent signal directly measure the reaction while amplification is taking place

3 Real Time PCR Chemistries
TaqMan Probe Molecular Beacon Scorpion Probe SYBR Green Amplifluor Direct Plexor LUX

4 TaqMan Probes Depend on Förster Resonance Energy Transfer (FRET) – inhibition of one dye caused by another without emission of a proton R – reporter Q- quencher The reporter dye is found on the 5’ end of the probe and the quencher at the 3’ end

5 TaqMan Probes These probes are designed to hybridize to an internal region of a PCR product In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe

6 TaqMan Probes Use DNA Polymerase to hydrolyse an oligonucleotide that is hybridized to the target amplicon Away from the quenching dye, the light emitted from the reporter dye in an excited state can now be observed

7 TaqMan Probes Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage Well-designed TaqMan probes require very little optimization can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair expensive to synthesize

8 TaqMan Probes

9 TaqMan MGB MGB – minor groove binder
shorter in length (12-15 nt) with chemical compounds attached to their 3' ends, which bind to the minor groove of the DNA Minor groove binding molecules are covalently bound to oligonucleotides which in their base sequence are complementary to a target sequence of single stranded or double stranded DNA The covalently bound oligonucleotide minor groove binder conjugates strongly bind to the target sequence of the complementary strand

10 TaqMan MGB MGB moiety gives greater stability to the hybridized probe, which raises its melting temperature during amplification making them better suited to applications such as SNP genotyping and other allelic discrimination applications MGB image

11 TaqMan LNA LNA – locked nucleic acid
type of nucleic acid analog that confers greater thermal stability to the probe, reducing background fluorescence and increasing the duplex melting temperature probes can be smaller, which is more effective in studying single nucleotide changes (such as with SNP genotyping analysis)

12 Molecular Beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure The loop contains a probe sequence that is complementary to a target sequence and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence A fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm

13 Molecular Beacons do not fluoresce when they are free in solution
when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly

14 Molecular Beacons added to the assay mixture before carrying out gene amplification and fluorescence is measured in real time provides an additional level of specificity can be synthesized that possess differently colored fluorophores, enabling assays to be carried out that simultaneously detect different targets in the same reaction can be expensive to synthesize, with a separate probe required for each target

15 Molecular Beacons easily discriminate target sequences that differ from one another by a single nucleotide substitution ideal probes for use in diagnostic assays designed for genetic screening, SNP detection, and pharmacogenetic applications

16 Molecular Beacons

17 Molecular Beacons have three key properties that enable the design of new and powerful diagnostic assays: they only fluoresce when bound to their targets, they can be labeled with a fluorophore of any desired color, and they are so specific that they easily discriminate single-nucleotide polymorphisms many as seven differently colored molecular beacons can be designed provide a new tool for increasing the effectiveness and lowering the cost of clinical diagnostic assays

18 Scorpion Probes sequence-specific priming and PCR product detection is achieved using a single oligonucleotide The Scorpion probe maintains a stem-loop configuration in the unhybridized state sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer

19 Scorpion Probes After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop This prevents the fluorescence from being quenched and a signal is observed confer very sensitive detection, as “when the sequence is a minority sequence in a background of a similar majority sequence, such as with mutation detections

20 Scorpion Probes

21 Scorpion Probes

22 SYBR Green provides the simplest and most economical format for detecting and quantitating PCR products in real-time reactions SYBR Green binds double-stranded DNA, and upon excitation emits light PCR product accumulates, fluorescence increases The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive

23 SYBR Green disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles.

24 SYBR Green Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed Good primer detection by SYBR Green requires extensive optimization Probe format animation

25 LUX LUX - light upon extension
does not use a probe, but label one of the primers with the fluorophore synthesize the labeled primer such that a hairpin structure is created this hairpin structure confers quenching of the fluorophore. primer anneals and extension begins, the primer becomes linear and fluorescence is released fluorophore is attached to the primer [instead of the probe], the fluorescence becomes part of the amplicon, allowing the use of melt (dissociation) curve analysis to verify specificity

26 LUX Melt curves cannot be used with most probe-based systems (e.g., TaqMan®) because the fluorescence is cleaved away from the amplicon LUX technology uses only two oligos per gene (forward and reverse primers) instead of three oligos (two primers plus probe) easier to design good multiplex assays that avoid cross-talk between the oligos

27 Application Probe format Application TaqMan General quantification
TaqMan MGB/LNA SNP detection, allelic discrimination Molecular beacon SNP detection, genetic screening, pharmacogenetic application Scorpion SNP detection, haplotyping, allelic discrimination SYBR Green

28 Comparison Probe format Advantages Disadvantages TaqMan
Specific, multiplex Expansive, different probes is required, quencher always fluoresces, TaqMan MGB Better quenching, Allelic discrimination May inhibit PCR Molecular beacon Multiplex (more common quenchers can be used) Scorpion Fast probing = higher signal, low background, more sensitive SYBR Green Cheap, easy to use, sensitive Non specific

29 Thank you