An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH

Transcription

1 An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH

2 DNA basic principles DNA carries the information of life on Earth DNA is present in all organisms (some viruses use RNA) DNA is a double stranded, linear, polymeric molecule built up of nucleosids forming an double helix structure The language of DNA is built up of four letters (bases A, G, C, T) These letters form words of three letters, each (genetic code) These words are translated into amino acid sequences the proteins The proteins carry functionality of life on Earth A G T C

3 In vivo DNA replication For proliferation of cells, DNA must be copied A set of enzymes is responsible for the copy process The principle is based on separating the complementary double strand and completing the single strands with complementary sequence to two new double strands The enzyme synthesizing the new strand is called DNA polymerase

4 PCR what is it? Kary Mullis: Invented PCR in 1983 Nobel Prize in 1993 PCR, or Polymerase Chain reaction is defined as a molecular technique which allows the production of large quantities of a specific DNA from a DNA template using a simple enzymatic reaction without a living organsim.

5 Conventional PCR what you need for it Buffer containing Mg 2+ A G T C specific Taq Components of PCR Mix are: - Template DNA with target sequence - Pair of sequence specific oligonucleotide primers - dntps - Taq DNA Polymerase - Buffer containing Mg 2+ Thermocycler

6 T[ C] Conventional PCR Step 1 Denaturation 96 C t[sec] Step 1: Denaturation of DNA double strand by Temperature (95 C) Result: (Two) single strands

7 T[ C] Conventional PCR Step 2 Annealing t[sec] 60 C Step 2: Annealing of Primers by lowering temperature (60 C) Result: Amplificable single strand with short double strand region where Polymerase can dock

8 T[ C] Conventional PCR Step 3 Elongation 72 C t[sec] Taq Taq Step 3: Elongation of primers by Taq-Polymerase at optimal temperature (72 C) Result: Two double strand copies of the target sequence

9 Conventional PCR repetition of temperature profile x x Repetition of the cyclus (temperature-time profile) leads to an exponential increase of target sequence copies

10 Conventional PCR interesting to know The complete PCR program has 3 main phases: 1. Initial denaturation 2. Repetition of the amplification cycle (3 temperature steps) 3. Cooling down modern PCR protocols have only one temperature step for annealing and elongation (Two-Step-PCR)

11 Conventional PCR detection by Gel electrophoreses Amplified DNA copies have an identical size The PCR products can be separated by size using agarose gel electrophoresis DNA can be visualized by a fluorescence dye and photo documentation

12 Conventional PCR the problems Work intensive and time consuming detection Gelelectrophoresis often needs handling of toxic ethidium bromide Limited sensitivity due to detection of gelelectrophoresis Problematic documentation of results by photography No good quantification possible Enormous risk of backward contamination since PCR tubes with high target sequence loads have to be opened for detection!

13 Real-time PCR Buffer containing Mg 2+ A G T C Taq Optical Unit FRET Thermocycler Unit Basically works like a conventional PCR Production of DNA copies is coupled with release of fluorescence (different systems for production of fluorescence) Fluorescence can be detected in the thermocyler, this enables a real-time monitoring of PCR progress

14 FRET: The fluorescence resonance energy transfer FRET Fluorescence dye I Fluorescence dye II (Quencher) Detector!!

15 Real-time PCR Hydrolysation-Probes (Taqman-principle) Probe FRET Primer A probe is an oligonucleotide with complementary sequence to an section of the target sequence The probe anneals to the DNA with the primers Attached to the probe is a fluorescence dye and a quencher inhibiting light emission of the fluorescence dye

16 Real-time PCR Hydrolysation-Probes (Taqman-principle)! Taq The probe is hydrolized by Taq polymerase 5-3 exonuclease activity Fluorescence dye and quencher are separated and fluroescence dye can develop fluorescence activity

17 Real-time PCR - all together now! Optical Unit fluorescence Ct 8 Ct 12 Ct 15 cycles Thermocycler Unit

18 real-time PCR devices and their technology Light source for exciting LED Laser Halogen Detector PMT CCD Photodiode PCR Vessels Glass Capillaries Plastic tubes Plastic plates with foils Optical Unit Thermocycler Peltier Air Thermocycler Unit Light of a defined wavelength is applied on the sample to excite fluorescence dye Fluorescing probes emit light of an alternative wavelength Emitted light is detected and processed by a computer

19 Real-time PCR devices Block Cycler Air Cycler Qiagen Rotor- Gene Q BioRad IQ5 BioRad myiq BioRad CFX348 ABI 7500 Corbett Rotor- Gene 6000 Roche LightCycler 2.0 ABI 7000 Roche LC480 Eppendorf realplex Corbett Rotor- Gene 3000 ABI 7700 Roche LightCycler 1.5 ABI 7900 ABI StepOne System Cepheid SmartCycler Agilent / Stratagene Mx3000p

20 Real-time PCR: The Advantages Short analysis time since detection is done during the amplification process Better data interpretation and documentation No backward contamination since tubes are not opened post PCR Increased specificity due to an additional specific detection molecule the probe More sensitive than conventional PCR with agarose gel detection No need to handle toxic ethidium bromide Quantification is possible!

21 Real-time PCR: Laboratory & Equipment Dr. F. Waldherr CONGEN Biotechnologie GmbH

22 General steps in real-time PCR analysis sample preparation enrichment and DNA extraction real-time PCR preparation real-time PCR and data interpretation

23 DNA extraction What you need Equipment for enrichment and homogenization (depending on matrix) Analytical balance Pipettes, filter tips SureFood PREP kit Thermomixer Centrifuge What you do Homogenization Pre-enrichment / enrichment Dosing Incubation / lysis DNA extraction and purification Prep kit

24 Real-time PCR (preparation, PCR and data interpretation) Separate pipettes and filter tips Separate gloves PCR work bench DNA work bench Respective plastics, foils SureFood real-time PCR kit Separate pipettes and filter tips Real-time PCR cycler Computer What you need realtime PCR cycler DNA What you do Master mix preparation Preparation of controls DNA addition Real-time PCR setup Real-time PCR Data processing interpretation Realtime PCR kit MM

25 Identification of critical points in laboratory process Real-time PCR is a sensitive method. So frequent sources of errors are: Cross-contamination of samples during homogenization due to contaminated homogenization tools Cross-contamination during DNA extraction due to contaminated pipettes Contamination of real-time PCR preparations due to contaminated pipettes During the whole process insertion of contamination in experiment by performing person (hands, clothes, ) Backward contamination due to opened PCR-tubes/capillaries (high DNA concentrations) By structuring laboratory setup and workflow a performance practical free of errors is possible!

26 The basic principle of one way street: Example for laboratory structure Flue PCR hood DNA Freezer (DNA) Laminar Flow Work Bench Incubator PCR hood Master mix DNA free Freezer (PCR)

27 Example for laboratory structure Sample DNA DNA M M realtime PCR cycler PREP kit DNA free M M MM RESULT Realtime PCR kit

28 Main rules for clean PCR Establish a one way workflow Use gloves and change them at critical points Use seperate pipettes (filtertips!) for different experimental steps Do locally separate Mastermix preparation and DNA Do use PCR work-benches Do not open PCR-vials/capillaries after real-time PCR-run Take care of disposal of used PCR-vials/capillaries Perform the required controls

29 Controls DNA-extraction Negative extraction control What is checked? The purity of the used extraction reagents and the correct performance and handling without cross contamination How to do: Make an extraction without any sample material Expected result: negative -? PREP kit Negative extraction control samples

30 Controls real-time PCR I Positive PCR control What is checked? correct performance of PCR How to do: add target DNA Expected result: positive Negative PCR control What is checked? possible contamination of PCR reagents How to do: No DNA addition Expected result: negative Buffer containing Mg 2+ Buffer containing Mg 2+ Taq Taq Positive control Negative control

31 Controls real-time PCR II PCR Inhibition control What is checked? presence of PCR inhibiting substances in sample DNA Problem: no standardized procedure ISO 24276: The PCR inhibition control may be used to demonstrate the absence of soluble inhibitors. Possible inhibition control systems Homologuos inhibition control (identical PCR system as for target) Heterologuos inhibition control (different PCR system than for target) External inhibition control (2 seperate PCR tubes) Internal inhibition control (1 PCR tube) Das SureFood PLUS System: heterologuos, internal inhibition control

32 External inhibition control detection system fw primer detection gene Buffer MgCl2 dntps Taq Polymerase External Amplification Control system Control target (PCR-product with a defined conc.) FAM TAMRA probe detection gene M M 1 M M 2 fw primer control system FAM TAMRA rev primer detection gene probe control system rev primer control system Two different master mixes necessary, but only one detection channel!!

33 SureFood PLUS System (e.g. SureFood PLANT PLUS) Detection system Puffer Taq Polymerase dntps System for internal amplification control fw primer detection sequence MgCl2 target-dna of internal amplification control fw primer inhibition control Probe for detection sequence rev primer detection sequence Probe for inhibition control Two different detection channels are required rev primer inhibition control

34 Interpreation of inhibition controls Data interpretation for SureFood PLUS kits detection gene inhibition control interpretation positive positive positive negative positive negative negative negative no interpretation possible positive negative positive If there is an inhibition you can try 1. Dilution of sample DNA 2. Change DNA extraction and purification protocoll

35 Thank you for listening!