Probe systems and mastermixes in real-time PCR allowing mismatch - - PowerPoint PPT Presentation

Probe systems and mastermixes in real-time PCR allowing mismatch tolerance and mismatch discrimination

Suzan D. Pas1

1Viroscience lab, Erasmus MC, Rotterdam, the Netherlands 2Affiliations

Taqman or hydrolysis probe mismatches

TaqMan Assay

90-95 °C 60-62 °C

Primer and Probe Hybridization, Extension Cleavage & detection

Denaturation

Taqman probes in general have a Tm of ±69°C therefore allowing 2-3 mismatches

Guidelines real time PCR design (I)

• Amplicons:
• · select the probe first and design the primers as close as possible to the probe

without overlapping it. Amplicons of 50 to 150 bp are strongly recommended.

· GC content within 30-80% · avoid homopolymers, this is especially true for guanine, where runs of four

• r more Gs should be avoided
• General Taqman Probes:

· Tm should be within 68°C to 70°C · no Gs on the 5’ end · select the strand that gives the probe more C than G bases

• Primers:

· Tm should be within 58°C to 60°C · the last 5 bases at the 3 prime end should have no more than two G's or C's

• · ideally there are no mismatches in the last 5 nt at the 3’side

· keep the annealing Tm of the primers as close as possible · select primer pairs with minimal number of potential primer dimers and

primer hairpins as possible

Guidelines real time PCR design (II)

Hybridization temperature

tgggaggttctctccagcactagcagg Length 27 nt GC content 60% Tm 69 ºC tgggaggttctctccagcactagcagg a t Tm 62.6 ºC

Tm = 81.5°C + 16.6°C x (log10[Na+] + [K+]) + 0.41°C x (%GC) – 675/N

tgggaggttctctccagcactagcagg a t a Tm 57.8 ºC

For calculation of Tm and impact of mismatches on Tm http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/

HIV-1 group M sequence variation in Gag and Pol genes

Emission Emission Excitation Excitation R Q R Q R – Long target-specific probe with fluor – Short quencher probe – Fluorescence quenched when probes are hybridized – Long probe preferentially binds target – Short quencher probe is dissociated – Fluorescence is detected

Novel probe technology: Partially double stranded linear DNA probes

Uncoupling of Detection and Extension Step

Key to Increased Mismatch Tolerance

TaqMan Assay

90

• 95

° C 50

• 60

° C

Extension, Probe Hybridization, Cleavage Denaturation

TaqMan Assay

95 ° C 60 ° C

Extension, Probe Hybridization, Cleavage & detection Denaturation

Abbott RealTi m e HIV

• 1

Abbott RealTi m e HIV

• 1

Luk et al. J Virol Methods 144;2007,1-11

Mismatch tolerance of partially double stranded probes

R Q

Target transcripts: 1 wt 16 mutant (2-6 mm)

Luk et al. J Virol Methods 144;2007,1-11

Impact of the length of target specific probe (P) on mismatch tolerance Impact of the length of quencher probe (Q) on mismatch tolerance

Meltcurve analysis

4.7 4 4.6 5 4.7 3 4.7 4 4.5 5 4.5 3 4.8 4 4.6 5 4.5 5 4.2 5 4.5 6 4.3 3 4.6 2 4.7 2 4.7 4 4.5 4 4.6

(mm) (log10)

4.7 4 4.6 5 4.7 3 4.7 4 4.5 5 4.5 3 4.8 4 4.6 5 4.5 5 4.2 5 4.5 6 4.3 3 4.6 2 4.7 2 4.7 4 4.5 4 4.6

(mm) (log10)

4.7 4 4.6 5 4.7 3 4.7 4 4.5 5 4.5 3 4.8 4 4.6 5 4.5 5 4.2 5 4.5 6 4.3 3 4.6 2 4.7 2 4.7 4 4.5 4 4.6

(mm) (log10)

High sensitivity and specificity with exceptional tolerance to nucleotide

• mismatches. Ideal for homogeneous detection of polymorphic targets.

Partially Double-Stranded linear Probe P45/Q11: High mismatch tolerance

Partially Double-Stranded Linear Probe Design Mismatches Discriminate OR Tolerate Quencher Conc High Low Quencher Tm High Low Target specific Probe Tm Low High Detection Temperature High Low F Q

Assays for the detection of single mutations

• Conventional Sanger sequencing: ~25-50%
• Sensitive methods:
• LIPA/DNA microarray 5-10%
• LigAmp 0.01-0.1%
• 454 sequencing 0.5-1%
• Digital PCR <0.001 - 10%
• Quantitative real-time techniques: 1-10%
• LNA/MGB probes (short high affinity probes)
• Molecular beacons
• Scorpions

Minor groove binding probes Locked nucleic acid probes

• Due to higher affinity binding shorter probes can be defined
• Taqman probes are 24-29 nt long
• LNA/MGB probes 14-19 nt long

Hybridization temperature: effect in MGB and LNA probes

Tm = 81.5°C + 16.6°C x (log10[Na+] + [K+]) + 0.41°C x (%GC) – 675/N

GGAGG(+T)T(+C)TCT(+C)CAG(+C)A

Length 17 nt GC content 59% Tm 69 ºC

GGAGG(+T)T(+C)TCT(+C)CAG(+C)A A

Tm 59 ºC

tgggaggttctctccagcactagcagg

Length 27 nt GC content 60% Tm 69 ºC

tgggaggttctctccagcactagcagg a t

Tm 62.6 ºC

tgggaggttctctccagcactagcagg a t a

Tm 57.8 ºC

Detection of oseltamivir resistant influenza A/H1N1 H274Y by real-time discrimination PCR using LNA probes

NTC + control + control NTC + control + control Erhard van der Vries et al., ESCV 2009

Wild-type cluster Mutant cluster

NA: 5’atcgaaaagggaaaggttactaaatcaatagagttaaatgcacccaattttCattatgaggaatgttcctgttacccagacactggc 3’ N1274Yfpr1 (30bp) N1274Yrpr1 (24bp) LNA:H274Y T (mut) LNA:H274H (16bp)

H/Y-PCR target

DFO FAM

Molecular beacon DNA probe

F

C G C A A A G T A T C A T C C C T C C A G G G C G G C C A A T T

Q

F

C G C A A A G T A T C A T C C C T C C A G G G C G G C C A A T T C G C A A A G T A T C A T C C C T C C A G G G C G G C C A A T T

Q

NASBA/PCR RNA

• r

DNA NASBA/PCR RNA

• r

DNA

C G T T T C A T A G T A G G G A G G T A C G T T T C A T A G T A G G G A G G T A C G T T T C A T A G T A G G G A G G T A

Molecular Beacons: mode of action

F

G C A A A G T A T C A T C C C T C C A G C G C C A A G G C G T T

Q

F

G C A A A G T A T C A T C C C T C C A G C G C C A A C G C C A A C G C C A A G G C G T T G G C G T T G G C G T T

Q

temperature

85 °C 25 °C

Mutation discrimination with molecular beacons

YMDD (atg) no template

Pas et al., Journal of Clinical Virology 32 (2005) 166–172

YSDD (atg)

x

YIDD (att)

x

Pas et al., Journal of Clinical Virology 32 (2005) 166–172

Influence of mastermix composition on primer bindingsite mismatch tolerance

Influence of mismatches in the primer binding site on Real-time (RT-)PCR efficiency

<10 fold

>10 and <100 fold

>100 fold under estimation!! Low impact, combined mismatches at 3’ end No amplification!!

Preventing underquantification due to mutations in primers and probes

• Avoid mutations in the last 5 nucleotides of the 3‘ part of the

primers

• Avoid C-C!!, A-A, G-G, G-A en A-G
• Avoid mutations in the last nucleotide at the 3‘ part of the

primers

• Avoid T-C, C-T en T-T
• A-C, C-A, T-G en G-T mismatches are relatively well

tolerated

Influence of mastermix composition on mismatch tolerance in the primer regions

MMLV / Taqgold combination (ABI): RT @ 48°C

Influence of mastermix composition on mismatch tolerance in the primer regions

rTTH based mastermix: RT@60°C

Mismatch effects at 3’-end position : DNA and RNA compared DNA: Taq gold polymerase RNA: MMLV / Taq gold polymerase

90

• 95

° C 50

• 60

° C

Extension, Probe Hybridization, Cleavage & Read Denaturation

95 ° C 60 ° C

Extension, Probe Hybridization, Cleavage & Read Denaturation 48 °C

Reverse transcription

Conclusions

• With highly variable targets like viruses, bacteria and variable regions

within eukaryotic genomes special attention should be paid to probe design and mastermix composition (rTtH versus Taq as polymerase, Glycerol/MgCl2/dNTP/ concentrations) Mismatch tolerance:

• Highly polymorphic target at rev primer use MMLV/Taqgold
• Highly polymorphic target at fwd primer use rTth based mastermix

Variant detection techniques

Fung, Antivir Ther 2004; Locarnini, Antivir Ther 2004

Time HBV Replication

Lipa 10% Sanger sequencing 25-50% Allele specific PCR 1-5% NGS 0.5%