Genetics II
LAB EXPERIMENT 5: PCR (Polymerase chain reaction)
Background & Theory
PCR is a very simple method used for the amplification of a specific DNA region (insert). Every PCR include the following steps: a) Initiation step – heating up the reaction to 94°C -96°C, b) DNA denaturation, c) primer annealing, d) extension or elongation step, e) final elongation and f) hold.
During the denaturation step the DNA molecule is being unwind to acquire a single chain DNA, used as a template for the DNA replication step with the help of DNA Taq polymerase enzyme. The next step involves the temperature decrease in order to create the adequate temperature for primers to bind to the DNA region. The DNA polymerase binds to the primer-template hybrid and begins DNA formation. During the elongation phase the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. The final elongation step is performed at a temperature of 70°C – 74°C for 2 to 15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. In the last step we keep our samples on hold, the temperature range is from4°Cto 15°Cfor an indefinite time for short-term storage of the reaction. Most PCR reaction has 20-40 cycles. Here, a basic, straight-forward PCR protocol is presented (Table 1).
Table 1: PCR set up and components
Components / Volume / PCR steps / Temperature / Duratıon / No. of cyclesddH2O / 17.8 μl / Initial Denaturation / 95°C / 2 minutes / 32 cycles
Taq polymerase buffer(10X) / 5 μl / Denaturation / 94°C / 30 seconds
MgCl2 (25 mM) / 3 μl / Annealing / 60°C / 1 minute
dNTPs (10mM) / 1 μl / Primer elongation / 70°C / 1 minute
Forward primer / 1 μl / Final elongation / 70 °C / 3 minutes / 1x
Reverse primer / 1 μl / Hold / 4°C / Forever / ∞
DNA template / 1 μl
Taq Polymerase / 0.2 μl
Total volume 30 μl
GENERAL GUIDELINES:
PCR machine:PCR MACHINE: THERMALCYCLER QPCR from Applied Biosystems
DNA Template:
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 μl reaction are as follows:
genomic / 1 ng–1 μg
plasmid or viral / 1 pg–1 ng
Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 ( can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05–1 μM, typically 0.1–0.5μM.
Mg++ and additives:
Mg2+ in general stabilizes primer-template complexes. If the Mg2+ concentration is too low, the yield of PCR product could be reduced. On the contrary, non-specific PCR products may appear and the PCR fidelity may be reduced if the Mg2+ concentration is too high.
Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNAPolymerase.
The final Mg++ concentration in 1X Standard TaqReaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2.
Deoxynucleotides:
The recommended concentration of each dNTP is 0.2 mM. In certain PCR applications higher dNTP concentrations are required. Due to the binding of Mg2+to dNTPs, Mg2+concentration needs to be adjusted accordingly. It is essential to have equal concentrations of all four nucleotides (dATP, dCTP, dGTP and dTTP). If the nucleotide concentrations are not balanced, the PCR error rate may dramatically increase. The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.
TaqDNA Polymerase Concentration:
Taq polymerase is a heat stable enzyme used in the polymerase chain reaction (PCR) to amplify segments of DNA in the lab. It was discovered in the heat-loving bacterium Thermusaquaticus, and without it, we couldn't amplify DNA! We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5units/50 μl reaction) in specialized applications.
One U is defined as the amount of the enzyme that produces a certain amount ofenzymatic activity, that is, the amount thatcatalyzesthe conversion of 1micro-moleofsubstrateper minute. The conditions also have to be specified: one usually takes a temperature of 25°C
Tips for OCR optimization:
Denaturation:
An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended.During thermocycling a 15–30 second denaturation at 95°C is recommended.
Annealing:
The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.Many online TM calculators are available for annealing temperature calculations .
Extension:
The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.In our case the
Cycle number:
Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.
Validating the Reaction
Once your PCR reaction has run, there are some ways of determining success or failure. The first is to simply take some of the final reaction and run it out on an agarose gel with an appropriate molecular weight marker to make sure that the reaction was successful and if the amplified product is the expected size relative to the maker.
The ultimate validation of a PCR reaction is to directly sequence the amplicon. This is often a choice that is not readily available since not everyone has access to a DNA sequencer nor will they have either the time or the funds to carry out such an analysis. One way to indirectly assess the sequence of an amplicon, however, is to carry out restriction enzyme digests on it. Given the vanishingly low likelihood that two well chosen primers will amplify an incorrect amplicon that matches the expected size, it is even more unlikely that an incorrect amplicon will give an expected pattern of restriction fragments.
References
Kainz P. (2000) The PCR plateau phase- towards an understanding of its limitations. BiochemBiophysActa, 1494: 23−27. For additional information, see the following resources for exploring the polymerase chain reaction and its variants.
Bustin SA. (2004) A to Z of Quantitative PCR. LaJolla, California: International University Line.
Chen B-Y, and Janes HW. (2002) PCR Cloning Protocols, Second Edition. Totowa, New Jersey: Humana Press.
Dieffenbach CW, and GS Dveksler. (2003) PCR Primer: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Date:08.12.2017
Prepared by: Senior assistant Jasmin Šutković