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Dr. Chaim Wachtel

Dr. Chaim Wachtel. Introduction to PCR and qPCR Part II: PCR!!. qPCR technical workflow. DNA Extraction. Data Analysis. Sampling. qPCR. RNA Extraction. DNase treatment. Reverse Transcription. Primer design. Primer design – key to successful PCR.

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Dr. Chaim Wachtel

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  1. Dr. Chaim Wachtel Introduction to PCR and qPCR Part II: PCR!!

  2. qPCR technical workflow DNA Extraction Data Analysis Sampling qPCR RNA Extraction DNase treatment Reverse Transcription

  3. Primer design

  4. Primer design – key to successful PCR Good primer design saves time and money Advanced applications require even more stringent primer design Multiplex Low abundance

  5. Good primer (pair) properties Primers should have 18-24 bases 40-60% G/C Balanced distribution of G/C and A/T bases Tm that allows annealing at 55-65°C No internal secondary structures (hair-pins) Primer pairs should have Similar melting temperatures, Tm , within 2-3 °C No significant complementarity (> 2-3 bp) particularly not in the 3’-ends

  6. The primer dimer (PD) problem Primers that interact are amplified by PCR. PD formation competes with the designed PCR and can compromise the reaction efficiency. Sense ´3 ´3 Antisense Cycling... Sense cAntisense Antisense Sense Sense Antisense

  7. Solution to the PD problem Reduce the formation of PDs by Good primer design (avoid 3’ complementarity) Minimal annealing time Good laboratory practice HotStart TouchDown Reduce the signal from PDs by Measuring fluorescence above the Tm of the PDs Use sequence-specific probe

  8. Considerations Avoid targets with secondary structure Avoid pseudogenes Avoid genomic contamination by designing primers to span intron-exon-junctions PCR primers introns exons

  9. Links for designing primers • http://www.tataa.com/ • http://www.ncbi.nlm.nih.gov/BLAST/ • www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html • www.ensembl.org • http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi • http://www.bioinfo.rpi.edu/applications/mfold/dna/form1.cgi • Primer Design- Beacon Designer/AlelleID • Primer express 3 (AB)

  10. Primer Express • Located on Software 1 • Easy to use • Not fool-proof, but none of them are…..

  11. Primer design-work flow No Satis- factory? Find sequence Design Primers Check Primers Yes Run PCR NCBI or Ensembl Primer3 or similar software for desired parameters - Tm - amplicon size • secondary structure • - complementarity - specificity Netprimer, BLAST and similar software …and gel electrophoresis to check specificity and functionality

  12. TaqMan Probe Design Amplicon size 70-150 bp Tm of probe 68-70 °C G/C content 30-70% No G at the 5´end Avoid runs of identical nucleotides Avoid secondary structure Avoid complementarity with primers HPLC purification

  13. Popular dyes and quenchers FAM JOE HEX TET VIC ROX Cy • DABCYL • TAMRA • Black Hole Quenchers

  14. RT-PCR • Housekeeping genes • What are they • How do you choose • Standard curve • Primer Dimer • Melt curve • Optimization • Test samples Reference

  15. Workflow – preliminary data analysis

  16. Baseline settings Baseline - is the initial cycles in PCR where there is little change in fluorecence signal, usually cycle ~3-15 • Set the baseline • Fixed number of cycles • Adaptive baseline • Control baselines in the linear scale (y-axis)

  17. Raw data

  18. Baseline adjustment

  19. The different phases Exponential growth phase Plateau phase Part of exponential growth phase where signal > background(noise) Samples must be compared in the exponential phase

  20. Setting threshold Purpose: Find a level of fluorescence where samples can be compared The theoreticalcycle where a sample intersect the threshold is called Ct Linear scale Logarithmic scale Threshold level Log Ct values

  21. Setting threshold Ct (threshold cycle): Threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number

  22. Setting threshold Several methods available for threshold setting Standard deviation of the noisefor the first few cycles Second derivative maximum (SDM) Best fit of standard curve (highest r2) Manual setting A two-fold difference in copy number should have one Ct difference no matter where the threshold is set within the exponential phase

  23. Dilution series and standard curves Used to control the quality of your assays Absolute quantification Standards = Diluted templates of known concentration Standard curve = Ct of each standard sample is plotted against the known concentration Used to determine concentrations of unknown samples Absolute quantification is dependent on the quality of the standard curve

  24. Standard curve Comment: Always cover the whole range of sample concentrations.

  25. Interpretation ofthe standard curve Linear regressionY = ax + ba = slope that gives efficiency of PCR from 10–1/a = 1 + efficiency b = # of cycles for detecting one molecule

  26. 30 25 20 15 10 5 0 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 Concentration (log scale)

  27. Relative quantification Often there is no good standard available Compare amount with reference Reference genes Genomic DNA Spike Ribosomal RNA Example Expression of target gene is 10% of the expr. of housekeeping gene. Same gene in other tissue, expression is 100%.

  28. Comparing treatments

  29. MIQUE Nomenclature • MIQUE - Minimum Information for Publication of Quantitative Real-Time PCR experiments Suggested nomenclature • Reference genes not housekeeping genes • Quantification not quantitation • Hydrolysis probes not TaqMan probes • Quantification cycle Cq replaces Ct, Cp, TOP

  30. Melting curve analysis Melting curves are obtained by measuring the fluorescence while increasing temperature Use a dye binding to double stranded DNA 70 95 Temp 80 90

  31. Melting curve analysis Confirms formation of the expected product (each dsDNA has its characteristic melting temp Tm) Distinguishes between specific PCR products and non-specific products (e.g. primer-dimers) High resolution melt – mutation and methylation analysis

  32. Melting curve derivative 1st Tm = 90 °C Melting temperature Tm is characteristic of the %GC, length and sequence. The product can be identified from the Tm. Tm = 81.5 °C

  33. 4-steps PCR 4-steps PCR can be used to eliminate primer-dimer signals 40 cycles

  34. Example – 4 steps PCR

  35. 100% efficiency 75% efficiency

  36. 100% efficiency 90% efficiency

  37. 80% efficiency 50% efficiency

  38. Reference primer efficiency

  39. RT-PCR –testing samples • ALWAYS perform melt curve • ALWAYS run negative controls • No RT • No template • Always Always Always run standard curve • Triplicate of each sample!!

  40. Requirements for RT-PCR Experiment • Always perform standard curve • All samples in triplicate • NTC control • No RT control • Prepare mix without cDNA; add this to each tube separately • Divide plate by gene and not sample • Do not need reference gene on every plate • Melt Curve • Check RNA- otherwise don’t bother with experiment • Do not rely on only 1 reference gene- check more than one per project • Every project is different! • Don’t be afraid to ask me questions, especially BEFORE starting the project.

  41. Digital PCR From Relative quantity to absolute quantity

  42. Commercially available machines Fluidigm QuantaSoft (Life Technologies)

  43. Rain Dance Bio Rad QX100

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