PCR settings, pitfalls and artefacts

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Adam Martin Jordan
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Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie.

1 De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie. PCR settings, pitfalls and artefacts Dr. Sci. Sabine Franke May 17th 2013 Molecular Biology and Cytometry Course SCK CEN - Mol

2 Overview Qualitative PCR Reagents Primers Thermocyclers Controls Nested PCR Quantitative PCR

3 Qualitative PCR

4 PCR optimization sufficient and specific amplification.

5 Problem Low yield or no amplification product Multiple, nonspecific amplification products

6 Check basic parameters template quality template quantity PCR primer design reverse transcription magnesium concentration cycling parameters reaction buffer composition enzyme concentration cycling parameters PCR additives Thermocyclers others

7 Template quality RNA quality and purity is crucial for success Extraction and storage of RNA important

8 Template quantity Avoid differences in RNA quantity

9 PCR primer design Length ranging from bases G+C content 40 60% Avoid internal secondary structure (primer dimers/ hairpins) Avoid three G or C nucleotides in a row near the 3 -end Ideally, both primers should anneal at the same temperature. Annealing temperature starting ~ 5 C below calculated Tm Purity of primers (desalted, HPLC, )

10 PCR primer design Nonspecific priming - Secondary products and lack of specificity Primer dimer formation Lower yields Lower sensitivity IDT,Eurogentec, Promega,. BLAST search; eventually other primer

11 Reverse transcription Different reverse transcriptases have significantly different efficiencies.

12 Reverse transcription One step RT One step RT PCR is performed sequentially in the same tube using the entire amount of cdna synthesis products as the PCR template. Two step RT Two step RT-PCR are performed sequentially, but only a portion of the cdna products is used as the template for PCR, which is performed in a separate tube.

13 Reverse transcription One or two step RT-PCR One-step RT-PCR more sensitive and requires less pipetting Two-step RT-PCR allows multiple PCRs from a single RT reaction to quantify multiple targets or perform replicate assays.

14 Reverse transcription amount of RNA RNA quality and purity can affect first-strand cdna synthesis efficiency RNA degradation -> efficientcy RT reaction

15 Magnesium concentration Effects of magnesium concentration on PCR amplification Magnesium is important cofactor to help stabilization of DNA structure.

16 Cycling parameters Number of cycles: cycles

17 Reaction buffer composition Choise of nucleotides

18 Enzyme concentration Concentration influences PCR reaction

19 PCR additives Has to be checked for each protocol (example DMSO)

20 Thermocyclers Calibration - thermocyclers can loose calibration over time Some blocks on thermocyclers do not support fast cycling Older thermocyclers need mineral oil layer over reactions

21 Others DNA contaminants (labo area) plastics

22 Problem: No Product Cloning Issues Wrong Sequence Non-Specific Thermocycler X Issue [MgCl2] XX Template/ X XX XX XX Primer Cycling X X XX Conditions Enzyme X XX XX Reagents XX

23 Troubleshooting Weak or no amplification: Smears above band Sensitivity problem increase # cycles increase time at step(s) increase quantity of template increase Taq concentration by 2X try different Taq (e.g. Faststart) increase MgCl2, primers, dntps decrease # cycles decrease concentration of reagent(s) Certified reference material cell line documentation and verification

24 Troubleshooting low weight bands: high weight bands decrease MgCl2 concentration decrease # cycles raise annealing temperature decrease primer concentration decrease dntps decrease Taq concentration

25 Prevent How? Bad sequence Contamination Contamination cross contamination freeze/thaw cycles Bubbles Perform a bioinformatic evaluation separate RNA area sample prep, mix set-up, PCR flow Add DNA or RNA to your wells last filter tips (aerosol-resistant) Make aliquots Centrifuge PCR plate before loading

26 Controls

27 Control requests Plasmids work as control templates but can have slightly different conditions compared to gdna or cdna If possible: Certified reference material (cell line documentation) negative control(s): check for contamination Run a positive control (a sample known to amplify well)

28 Control Include an internal standard in your PCRs second primer pair that amplifies a housekeeping gene can be included in the reaction Participating in external QC rounds

29 Nested PCR

30 Nested PCR

31 Attention Special care for nested PCR! More sensitive, but opening increases the risk of contaminating subsequent reactions with amplified product. Never opening more than one tube at a time Using a separate thermocycler Adding additional negative control

32 Quantitative PCR

33 De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie. Quantitatif PCR design Quantif. samples analysis assays guidelines Ref.gene

34 Quantitative PCR Include an internal standard in RT-PCRs. housekeeping gene Housekeeping genes can help account for differences in the amounts of starting nucleic acid.

35 Tips Change positions of controls

36 Tips Pipette calibration frequently

37 Tips Do RNA extraction in one serie Do RT step in one serie

38 Tips Participating in QC rounds not only for tests but also for extraction/storage conditions

39 Housekeeping gene Include an internal standard in RT-PCRs. For example, a second primer pair that amplifies a housekeeping gene can be included in the reaction. (constant expression levels among the samples compared)

40 Housekeeping gene Stable expression Should be expressed in all cells HK gene should have the same copy number in all cells Transcription level in same range as target gene Perfect standard does not exist

41 Gene expression Gene expression result: impact of RNA quality Accuracy of gene expression is highly dependent onmrna quality Reference gene expression stability is influenced by RNA quality Quantitative RT-PCR partially degraded RNA may not give accurate representation of gene expression

42 Standard-curve method Absolute and relative quantification Interpretation/quantification outside standard curve

43 Efficienty Control the efficientcy of the reaction Variables which can affect the efficiency: - Length of the amplicon - Secondary structure - Primer design

44 Conclusion check working area before starting check primers before starting check protocol before starting check settings (cycles, baseline, threshold) Before starting use controls analyse problems step for step

45 Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention

Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design

Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design The Polymerase Chain Reaction (PCR) is a powerful technique used for the amplification of a specific segment of a nucleic acid