The Polymerase Chain Reaction (PCR) enables a powerful amplification of a specific target sequence – if it is present in the tested sample. PCR is used for many applications in life science research as well as clinical diagnostics.
In life science research, PCR is used for two very different types of applications – PCR is both used for sensitive detection purposes and as a "toolbox" method in different aspects of molecular biology applications, where requirements as to the level of sensitivity are very relaxed.
So, PCR can be viewed both as a sensitive diagnostic technology and a molecular biology toolbox method with no specific sensitivity requirements.
When performing the "insensitive" variant of PCR (initial copy number of target is above 100,000 in the reaction) – there are no specific requirements as to assay optimization or laboratory design. The initial copy number is so large that any standard buffer / enzyme will produce a positive result, as long as the primer design results in operational primers. Furthermore, The initial copy number "vaccinates" the assay against impact of carry-over contaminating PCR products from previous assays. Thus, there are no specific requirements as to laboratory design.
However, when performing the sensitive variant of PCR, all aspects of the assay must be optimized in order to ensure maximum sensitivity with minimal background and no contamination.
TINA-modified PCR primers will improve the efficacy (allowing shorter PCR programs, higher annealing temperatures, lower primer concentrations or higher stringency of the buffer system) of your Real-time PCR assay.
If you want to upgrade an existing Real-time PCR assay with TINA- primers, reorder the primers with a 5' TINA modification or if you are designing a new PCR assay, order the primers with a 5' TINA modification. The PCR conditions with TINA-primers has to be optimized (protocol points 5 to 8 below). Once you have identified the optimal combination of annealing temperature (Ta) and Mg⁺⁺, enzyme and primer concentrations in your buffer of choice, your “Optimal combination” implies that you cannot reduce your primer concentration or increase your Ta further without loss of efficiency and a raise in cycle of quantification.
IMPORTANT: If you are unable to observe this effect, your assay is un-stressed and must be optimized further. Following this optimization, you will be able to observe even lower primer concentrations and/or higher Ta by TINA –primers without compromising your assay efficacy.
If you are designing a new PCR assay and wish to take advantage of the increased efficacy made possible by TINA-primers, please observe the protocol below:
- Choose your real-time PCR platform
- Choose a reporter system - e.g. SYBR Green I (no internal probe) or TaqMan (internal probe)
- Design your probes AND THEREAFTER your PCR primers. Use conventional primer design software - e.g. Primer3
- Order your PCR primers with a 5' TINA modification here
- Choose your buffer. A stringent, low salt buffer should be used. The following buffers have been successfully tested in TINA-PCR: Qiagen QuantiTect SYBR Green, Roche FastStart SYBR Green, Invitrogen Power SYBR Green, TaKaRa SYBR Premix, KAPA SYBR FAST, Fischer Absolute qPCR SYBR Green, Agilent Brilliant III Ultra-Fast SYBR Green plus a “Home-made” 10x buffer (104 mM Tris-HCl, 568 mM Trizma-base, 160 mM (NH₄)₂SO₄, 0.1% Tween 80, 300 mM NaCl)
- Choose your enzyme (if not included in buffer system). The following polymerases have been successfully tested in TINA-PCR: Qiagen HotStarTaq, Roche FastStart Taq, Invitrogen AmpliTaq Gold, TaKaRa Ex Taq HS, KAPA SYBR, Fischer Thermo-Start plus Agilent Taq
- Optimize your Ta: Use positive control material, your buffer and enzyme of choice and 200 nM TINA-primers. Test Ta in the range from calculated Ta (e.g. Primer3) to the level where the PCR signal disappears. Please remember that the TINA modification increases the primer Ta around 3 to 4°C compared to the calculated Ta for conventional DNA primers. Evaluate the PCR amplification by cycle of quantification and choose Ta where amplification efficiency starts to decrease (cycle of quantification raises). For new protocols, please check that you get the expected amplicon as e.g. band on a gel. NB: Ta differs between PCR platforms – always re-optimize Ta when performing assay on new platform – or in a new reaction volume
- Optimize your primer concentration and Mg++ concentration at the Ta established previously: Test all possible combinations of: Primer (starting at 50 - 100 - 200 - 400 nM) and Mg++ (2 - 2.5 - 3 - 3.5 mM). Evaluate real-time PCR curves and identify optimal combinations of the variables and if necessary titrate the amount of polymerase used for each reaction
- Define analytical sensitivity by testing a serial dilution of a): positive control with known target copy number or b): other standardized positive control – using variables as defined using optimized Ta, primer concentrations and Mg++ concentrations
- Optional: Optimize PCR program cycling parameters (length of denaturing / annealing / elongation phase – number of cycles).