Monitoring PCR Amplifications using Sentinel TM Molecular Beacons.

Molecular Beacons : Structure Sanjay Tyagi and Fred Kramer (1996) Nature Biotechnology 14:

Molecular Beacons: Conformational Change

Fluorescence profile: PCR

Molecular Beacons Quenching Mechanism

Thermal Stabilities of Molecular Beacon hybrids and Linear Probe hybrids. Tyagi, S. et al. (1998) Nature Biotechnology 16: 49-53

Superior Ability to Discriminate Single Nucleotide Mismatches Mismatched hybrids formed by hairpin probes are less stable than mismatched hybrids formed by linear probes. Results in improved ability to discriminate single nucleotide mismatches. Especially important in allele discrimination assays. Enables multiple probe/target hybrids in a single tube to be discriminated more easily.

Molecular Beacons Cycling Parameters Mechanism does not impose limitations of 2 step PCR –limited annealing temperatures –sub-optimal polymerization 3 step PCR –more flexible design parameters –wide range of possible annealing temperatures –optimal polymerization temperature

Molecular Beacon Design Process: Summary Melting Curves Verify Melting temepatures experimentally Loop sequence Tm 7 to 10 o C higher than the desired annealing temperature Arm Sequences Add complementary arms that melt 7 to 10 o C higher than the desired annealing temperature 5 bp arms melt between 55 and 60 o C 6 bp arms melt between 60 and 65 o C 7 bp arms melt between 65 and 70 o C DNA folding Subject complete molecular beacon to a folding program to check for unwanted secondary structures ( Molecular Beacon Purity Signal to Background Ratio Determination Synthesis PCR primers Design primers to produce a relatively small amplicon, <150bp Check probe and primers for unwanted regions of complementarity Melting Curves Verify melting temperatures experimentally

Advantages of Molecular Beacons Probe confers high specificity of target detection Improved ability to detect single nucleotide mismatches for allele discrimination Non-FRET mechanism and universal quencher allows greater flexibility in fluorophore choices Greater cycling flexibility and optimized parameters

Molecular Beacons Applications Allelic discrimination Gene expression analysis –PCR –cells Infectious disease investigation –pathogen identification –viral load determination Environmental analysis –pathogen detection & quantification Food diagnostics –pathogen detection & quantification

Sentinel TM Molecular Beacon System Gene detection –determination of gene presence or absence using gene specific primers and molecular beacon Gene expression analysis –detection and quantification of mRNA using specific primers and molecular beacon Allele discrimination –detection of wild type & mutant alleles using gene- specific primers and allele-specific molecular beacons in a single tube format

1-tube RT-PCR core reagent kit RNA DNA PCR RT-PCR Cycler/detector 2-tube RT-PCR core reagent kit PCR core reagent kit cDNA synthesis Amplification Plot

Sentinel TM Molecular Beacon PCR Core Reagent Kit Quantification of mouse muscle nicotinic acetylcholine receptor g subunit DNA. Linearity of the molecular beacon detection.

Quantitation of Plasmid DNA After Transient Transfection (Relative to  -actin)

Sentinel TM Molecular Beacon 1- and 2-Tube RT-PCR Core Reagent Kits Comparison of 1-tube and 2-tube RT-PCR core reagent kits in relative gene quantitation.

Sentinel TM Molecular Beacon 1-Tube RT-PCR Core Reagent Kit Multiplex RT-PCR using human total RNA as target: Detection of GAPDH mRNA. Detection of HPRT mRNA.

Sentinel TM Molecular Beacon 1-Tube RT-PCR Core Reagent Kit 1-tube RT-PCR with human total RNA, detected with mRNA- specific  -actin molecular beacon. End-point acquisition of triplicate samples on the FluorTracker.

Sentinel TM Molecular Beacon 1-Tube RT-PCR Core Reagent Kit Intra-assay variability using the 1-tube RT-PCR core reagent kit and the GAPDH mRNA specific molecular beacon.

W/W W/MM/M PCR Allele Discrimination Assay

Allele Discrimination Kits genemutationprotein function CCR5 32bp deletion truncated Chemokine receptor Delays AIDS SDF1 (stromal-derived Factor-1) G to A substitution in 3’ untranslated ChemokineDelay or accelerate AIDS result CCR2 G to A substitution Val Ile Chemokine receptor Delays AIDS MTHFR (Methylene- tetrahydrofolate reductase) C to T substitution Ala Val High risk cardiovascular diseases Key enzyme in homocysteine metabolism Factor V G to A substitution Arg Gln 7x higher risk for vein thrombosis Inactivator of an anticoagulant

FluorTracker TM : End point Analysis 96-well fluorescence reader for PCR plates & tubes Reads PCR tubes directly w/o transfer of samples or removal of caps Separate thermal cycling and detection –Increases through-put: one reader for multiple cyclers Multiple fluorophore capability: Xenon flash lamp excitation source ( nm); ex/em controlled by filters (e.g. 485/520; 584/612) Allelic discrimination Determine presence or absence of specific target

DNA Sample End Point Fluorescence CCR2 mutation analysis on genomic DNA

Sentinel TM Molecular Beacon Detection Kits Genes: –Endogenous controls; GAPDH &  -actin –Recognize gDNA and cDNA (primers located within exons) –Optimized for human but will also recognize product amplified from mouse and rat templates Uses: –Endogenous control for detection of PCR inhibitors or PCR failures –Quantification of gDNA –Control for evaluation of newly designed molecular beacon systems

Sentinel TM Molecular Beacon  -Actin Detection Kit: Template Specificity

Human gDNA dilution series:GAPDH detection kit Template Amount (ng) Ct Molecular Beacon GAPDH Detection System Ct inversely  log template amount

Sentinel TM Molecular Beacon Expression Analysis Kits. Gene expression systems –endogenous controls for relative gene expression:  -actin (h or m), GAPDH (h),  -2-microglobulin (h) –specific targets: TNF  (h), aP2 (h), HPRT (h) High degree of specificity for mRNA Quantitation based on threshold cycle (Ct) –Linear over wide range of template amounts (>6 orders of magnitude dynamic range)

GAPDH Expression Analysis Target Specificity Sentinel Molecular Beacon GAPDH Expression Analysis TaqMan GAPDH Expression Analysis

Quantification of Human cDNA: GAPDH Expression Analysis

Induction of TNF  by LPS in THP1 Cells

Quantifying Gene Expression Using Molecular Beacons 5: differentiated/ lps 2: undifferentiated/lps 6: differentiated/IFNg 3: undifferentiated/IFNg human TNF 

Quantifying Gene Expression Using Molecular Beacons human aP differentiated undifferentiated

Sentinel TM Molecular Beacons: Summary Multiple systems that exploit advantages of molecular beacons technology Qualitative and Quantitative Analysis –Detection:  -actin and GAPDH –Expression Analysis:  -actin (h & m), GAPDH (h),  -2-microglobulin (h), TNF  (h), aP-2 (h), HPRT(h) –Core Reagent: PCR, 1-tube & 2-tube RT-PCR –Allelic discrimination –Instrumentation: FluorTracker, Spectrofluorometric thermal cycler

Acknowledgements Sanjay Tyagi Fred Kramer Michelle Cayouette Anita Sucharczuk Taylor Reid Elizabeth Nash Jim Patnode Ali Mousavi Roger Taylor Reinhold Mueller Gothami Padmabandu Doug McKenzie Danny Hoang Brenda Rogers XiuYuan Hu Lingyu Chen

Sentinel TM Molecular Beacons for Monitoring PCR