Real-Time PCR Molecular biology methods for pharmacists Doc. RNDr. Jan Hosek, Ph.D. hosek@mail.muni.cz Department of Molecular Pharmacy FaF MU Phases of PCR amplification / 95 dag ^ +Primere I DNA Synthesis V Theoretically, the amount of the products is doubled during each PCR cycle > Really the doubling the PCR products is only going near to 100%. IOOOO ..................... ID 20 30 Cycle Number The basic principles of the method > Visualisation of growing amount of the products of amplification measured by growing fluorescence during PCR > Intensity of fluorescence correlates to the concentration of the template > The correlation between amount of the PCR products and intensity of fluorescence is used to calculation of the templates number at the beginning of PCR Real Time termocyclers Real-time PCR advantages The same or higher sensitivity without manipulation with samples — lower contamination risk Analysis without electrophoresis Automatization of process Sample quantification — the level of mRNA, amount of pathogen Possibility to run „multiplex" reactions Important components for performing Real Time PCR > Fluorophores > Quenchers > Probes Fluorophores > Mostly heterocyclic polyaromatic carbohydrates > Their terminal fluorescence (emission) depends on ability of fluorophore to absorb and to emit photons > Emission of the fluorophore strongly depends on temperature The principles of fluorescence Absorption of light with specific wavelength by fluorophore Exiting the fluorophore to a state with higher energy Return the molecule to the basic state which is followed by emission of photon with lower wavelength 1,0 & 0.6 □ 5 0.4 I 0.2 0.0 Exctatbn Emission ■ I ■ I I I■ I j I 111 ■ Vi Iv11v I iVt 11 I j I I i I I I I I j I I 300 350 400 450 500 550 Wavelength 600 650 700 Quenchers > Molecules which are able to absorb or dissipate energy from excited fluorophore > The quencher receives the energy from fluorophore and absorbs or dissipate it by the mechanism of „Proximal quenching" or „Fluorescence resonance energy transfer (FRET)" Proximal quenching > Based on short distance between fluorophore and quencher > This distance enables an effective transfer of energy which is transfer by quencher to warm and quenches the excited fluorophore by this Proximal quenching Fluorescence resonance energy transfer (FRET) > A donor molecule (excited by external light source) transfer a part of its energy to acceptor molecule which emits light with another wave length > Efficiency of this process depends also on distance of donor and acceptor molecules (effective about 100A, approx. 30 bp in linear probes) Fluorescence resonance energy transfer (FRET) Probe Short oligonucleotide with the similar features as PCR primer (probe binds to DNA template by the same manner) It enables to bind fluorophore and quencher in the effective distance and ensures the process of quenching the fluorophore Fluorophore, probe, and quencher - TTC ATG CTT GGA CCG GGA CTG ATT CCT - 5' 3 03HQJ) TTC ATG CTT GGA CCG - - TTC ATG CTT GGA CCG The most frequently used combinations of fluorophore/quencher and probe 250 300 350 400 450 500 550 600 650 700 750 Wavelength (nm) (^HQj) Formats of Real Time PCR i. Nonspecific formats: based on nonspecific binding of fluorophore into synthesized dsDNA molecule ii. Specific formats: based on specific binding of probe labelled by fluorophore Nonspecific formats - DNA intercalators - > Fluorofore intercalates to synthesized molecule of dsDNA during PCR > Binding into minor groove Formates > Quencher-Labeled Primer I > Quencher-Labeled Primer II > LUX™ Primers > Arrmlifluor™ c ■ ^ > SYBR Green I Principle of using of SYBR™ Green I Is binding of SYBR Green to dsDNA reversible or irreversible, and why? Detection of differences in DNA sequence based on SYBR Green I - model example - DNA sequence A that is 200 bp long cctcctgcctctaccaatcgccagtcaggaaggcagcctaccccgctg actccacctttgagagacactcatcctcaggccatgcagtggaattcc acaaccttccaccaaactctgcaagatcccagagtgagaggcctgtat ctccctgctggtggctccagttcaggaacagtaaaccctgttccgact actgcctc DNA sequence Ai with 5 bp insertion - total length 205 bp cctcctgcctctaccaatcgccagtcaggaaggcagcctaccccgctg actccacctttgagag^c4c7acactcatcctcaggccatgcagtgga attccacaaccttccaccaaactctgcaagatcccagagtgagaggcc tgtatctccctgctggtggctccagttcaggaacagtaaaccctgttcc gactactgcctc Result of detection by SYBR Green I - the basic data - Number of PCR cycles Result of detection by SYBR Green I - melting analysis - Am pi icon melting Fl uorescence g I X wt/wt \ Wt/Mut \ Mut/Mut -dF/dT Temperature X Wt/Wt X Wt/Mut \ MutyMut Temperature Differentiation by melting curve shape http: / / www.gene-quantification, de / hrm. html Specific formats - labelled DNA probes - Method based on primer annealing and hybridisation of probe to specific DNA locus Two basic probes exist > Linear probes > Structural probes Specific formats Linear probes > ResonSense® Probes > Angler® Probes > HyBeacons™ > Light-up Probes > Hydrolysis (TaqMan®) Probes > Lanthanide Probes > Hybridization Probes (FRET) > Eclipse™ > Displacement Hybridization/Complex Probe Structural probes > Molecular Beacons > Scorpions™ > Cyclicons™ > Nanoparticle Probes > Conjugated Polymers/Peptide Nucleic Acid Probes Linear probes Hydrolysis (TaqMan®) Probes 111111111111111111111111111111 ............................ I I I I I .............................. Linear probes Hydrolysis (TaqManf®) Probes u> Fluorophore Q Quencher Excitation Quenching I orward primer q u r _ laqMan probe y ^^^^^^^^^^^^^^^^^^^^^^^^^ 5' o Excitatio Allele specific TaqMan probes There are 2 probes bearing 2 different flourophores Each probe detects particular allele Allele n Mcmalcli Allele 2 V^/_INJ (9) II II 11.11 .11 II 11 Match Mk (Mi h FAM*<*» -srar http://www.ucl.ac.uk/ Fluorescence Fluorescence DNA quantification 1. Measuring Ct (threshold cycle) 2. Calibration curve formation 3. Quantification of unknown sample using the calibration curve Threshold cycle 4 > Ct - PCR cycle in 3.5 which the first change of fluorescence is detected § 2.5 > Ct is measured by «2 termocycler g ^ automatically E 1 0.5 0 -•-Sample —Ct Wu ■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■ r^lIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 2 12 22 32 42 Number of PCR cycles Calibration curve 4 3.5 3 a> £ 2.5 a> o t/> 9 CD ^ O 2 1.5 u_ 1 0.5 0 -*-10exp4 -■-10exp3 -a-10exp2 -*-10exp1 6 16 26 36 46 Number of PCR cycles 10 100 1000 Concentration 10000 Real picture of calibration curve ( Plot Settings \ Plot Type: ARn vs Cycle v Graph Type: Log v Color: Well Amplification Plot 0.01 § 0.001 0.0001 0,00001 0.000001 0.0000001 Legend A BlICl D I IE |F » ^ ü 36 38 40 42 ^ Plot Settings \ Target All v Plot Color Default Is/ E: Standard Curve 100 200 1000 Quantity Tarnet: Target 1 Slope: -3,568 y-lnter: 39,671 r2: 0,98 Eff%: 90,675 Legend - | Standard | Unknown | Unknown (Flagged) Quantitation of unknown sample 0 £35 u 2 30 % 20 4 3.5 a> 3 o g2.5 o (0 9 g> ^ § 1.5 E 1 0.5 0 40 -I 10 16 26 36 Number of PCR cycles 46 100 1000 Concentration 10000 Sample Type Ct Concentration (copies/i 1 Unknown 25,64 3,50E+03 2 Unknown 29,23 2,50E+02 K1 Standard 23,97 1,00E+04 K3 Standard 27,16 1,00E+03 K3 Standard 30,68 1,00E+02 K4 Standard 33,53 1,00E+01 Linear probes Hybridization Probes (FRET) j_u_u_ujj. jjjjjjjjj. FITC RED RED 7^ Using FRET analysis for detection of SNP - model example - Standard allele 200 bp long cctcctgcctctaccaatcgccagtcaggaaggcagcctaccccgctg actccacctttgagagacactcatcctcaggccatgcagtggaattcc acaaccttccaccaaactctgcaagatcccagagtgagaggcctgtat ctccctgctggtggctccagttcaggaacagtaaaccctgttccgact actgcctc Mutant allele (with SNP) 200 bp long cctcctgcctctaccaatcgccagtcaggaaggcagcctaccccgctg actccacctttgagagccactacactcatcctcaggccatgcagtgga attccacaaccttccaccaaactctgcaagatcccagagtgagaggcc tgtatctccctgctggtggctccagttcaggaacagtaaaccctgttcc gactactgcctc Design of probes for detection of SNP by FRET GAGAGATCACTCAT-FITC RED-CCATGCAGTGGA GACTCCACCTTTGAGAGATCACTCAfOTCCTCAGGCCATGCAGTGGA Principle of SNP analysis based of FRET probes Mutant allele (dCTP) Tm= 55°C ......... 1 1 1 1 RED ■ ■ ■ ■ Standard allele (dATP) Tm= 62°C 1 1 1 1 ..... ■ Ill J III! i i i i FITC RED Result of detection SNP by FRET - the basic data - 0 8 16 24 32 40 48 Number of PCR cycles Result of detection SNP by FRET - melting analysis - 4 T mutant homozygote (dCTP) standard homozygote (dATP) heterozygote (dATP/dCTP) 40 45 50 55 60 65 70 75 80 85 Temperature Structure probes - molecular beacon ■ Contains: ■ Loop with target complementary sequence ■ Stem which „closes" the hairpin ■ Reporter and quencher ■ High sensitivity — protects probe during reaction ■ SNPs detection ■ Allelic discrimination Loop Sequence Stem Sequence Loop Sequence 5' Reporter y 2 3* Quencher Q R 5' Reporter Amplified Target DNA 1. Unbound beacon with 2. Bound beacon with quenched fluorescence unquenched fluorescence Structure probes - Scorpions ■ Bi-function molecules — contain primer and probe in one molecule ■ The signal is detected one cycle after probe binding Digital PCR Theorecically - One DNA molecule in one reaction well Molecules in wells follow Poisson distribution => gDI pla< Wild type Mutant B © O © O O o o o © o o © o o o o o QOO oo© ©oo ©OO o©o Source: (Sedláka & Keith, 2013).10 Digital PCR Theorecically - One DNA molecule in one reaction well Molecules in wells follow Poisson distribution => „ cleaning" data by mathematical operations Result of the reaction is 1 or 0 (positive or negative) Preparation Distribution PCR reaction Readout gDNA, cDNA, RNA, plasma oooo Sample partitioned into many reactions •OOÖ •880 Positive reactions Negative reactions as 00 Absolute quantification http://www.lifetechnologies.com/ Digital PCR Preparation Distribution PCR reaction Readout gDNA, cDNA, RNA, plasma v ©ooo Sample partitioned into many reactions oooc 0S08 • Positive reactions O Negative reactions as KBBI Absolute quantification http://www.lifetechnologies.com/ Application of qPCR for ELISA How does it work? ProQuantum immunoassays utilize proximity ligation assay fPLA'") technology to combine antigen-antibody binding tor analyte detection with qPCR signal amplification and readout (Figure 1). The assay is a two-step process: A. Analyte binding by paired antibodies conjugated to oligonucleotides Two antibody conjugates are provided in each kit: a 3' end oligonucleotide and a 5' end oligonucleotide, each conjugated to a target-specific antibody. When the antibody pair binds to two different epitopes of the protein, the 3' and 5' oligos come into close proximity. B. Ligation of the oligonucleotides by DNA ligase and amplification by Applied Biosystems" TaqMane q PCR Assay Only when the pair of antibodies binds to the analyte (A) can the associated oligos become bound to the complementary splint oligo and subseqently joined to each other with DNA ligase (B). Following the oligo ligation, 95°C heat inactivation denatures the ligase. antibodies, and other proteins, leaving 100-base strands in concentrations proportional to the level of antibody-analyte binding in the first stage. This 100-base DNA strand serves as the amplification template for 40 cycles of qPCR using TaqMan Assays,