During the Pcr Activity, What Was the 1st Item You Added to Your Pcr Tube?

• Periodical List
• J Vis Exp
• (63); 2012
• PMC4846334

J Vis Exp. 2012; (63): 3998.

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Todd C. Lorenz

ⁱMicrobiology, Immunology, and Molecular Genetics, University of California, Los Angeles

Abstract

In the biological sciences there have been technological advances that catapult the subject area into golden ages of discovery. For example, the field of microbiology was transformed with the appearance of Anton van Leeuwenhoek'southward microscope, which allowed scientists to visualize prokaryotes for the beginning time. The evolution of the polymerase chain reaction (PCR) is ane of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biological science. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable Deoxyribonucleic acid polymerase from the bacterium Thermus aquaticus, consequently the proper noun Taq Deoxyribonucleic acid polymerase.

PCR is a powerful amplification technique that can generate an ample supply of a specific segment of Deoxyribonucleic acid (i.east., an amplicon) from only a minor corporeality of starting textile (i.east., DNA template or target sequence). While straightforward and generally problem-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific Dna products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Some other potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and conscientious troubleshooting are employed to sort out and solve the problem(southward). This protocol outlines the basic principles of PCR, provides a methodology that volition result in amplification of about target sequences, and presents strategies for optimizing a reaction. By post-obit this PCR guide, students should be able to: ● Prepare reactions and thermal cycling weather for a conventional PCR experiment ● Understand the function of diverse reaction components and their overall issue on a PCR experiment ● Design and optimize a PCR experiment for whatsoever DNA template ● Troubleshoot failed PCR experiments

Keywords: Bones Protocols, Outcome 63, PCR, optimization, primer design, melting temperature, T_m, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics

Protocol

1. Designing Primers

Designing appropriate primers is essential to the successful issue of a PCR experiment. When designing a gear up of primers to a specific region of Deoxyribonucleic acid desired for amplification, one primer should amalgamate to the plus strand, which by convention is oriented in the v' → iii' direction (also known as the sense or nontemplate strand) and the other primer should complement the minus strand, which is oriented in the three' → 5' direction (antisense or template strand). There are a few common bug that arise when designing primers: i) self-annealing of primers resulting in germination of secondary structures such as hairpin loops (Figure 1a); 2) primer annealing to each other, rather then the Dna template, creating primer dimers (Figure 1b); 3) drastically different melting temperatures (T_grand) for each primer, making it difficult to select an annealing temperature that will allow both primers to efficiently demark to their target sequence during themal cycling (Figure 1c) (See the sections Calculating MELTING TEMPERATURE (T_m) and MODIFICATIONS TO CYCLING Conditions for more than information on T_ms).

1. Below is a list of characteristics that should be considered when designing primers.

   1. Primer length should be 15-30 nucleotide residues (bases).

   2. Optimal Thou-C content should range between 40-60%.

   3. The three' end of primers should comprise a 1000 or C in guild to clamp the primer and prevent "breathing" of ends, increasing priming efficiency. Dna "breathing" occurs when ends practice not stay annealed but fray or split apart. The three hydrogen bonds in GC pairs aid prevent breathing but also increase the melting temperature of the primers.

   4. The iii' ends of a primer set, which includes a plus strand primer and a minus strand primer, should not be complementary to each other, nor tin can the 3' finish of a single primer exist complementary to other sequences in the primer. These two scenarios upshot in germination of primer dimers and hairpin loop structures, respectively.

   5. Optimal melting temperatures (T_m) for primers range between 52-58 °C, although the range tin be expanded to 45-65 °C. The final T_grand for both primers should differ past no more than than 5 °C.

   6. Di-nucleotide repeats (e.g., GCGCGCGCGC or ATATATATAT) or unmarried base runs (e.g., AAAAA or CCCCC) should exist avoided as they tin can cause slipping along the primed segment of Deoxyribonucleic acid and or hairpin loop structures to form. If unavoidable due to nature of the Deoxyribonucleic acid template, then only include repeats or unmarried base runs with a maximum of 4 bases.

Notes:

1. There are many calculator programs designed to aid in designing primer pairs. NCBI Primer blueprint tool http://www.ncbi.nlm.nih.gov/tools/primer-boom/ and Primer3 http://frodo.wi.mit.edu/primer3/ are recommended websites for this purpose.

2. In order to avert amplification of related pseudogenes or homologs information technology could exist useful to run a blast on NCBI to check for the target specificity of the primers.

ii. Materials and Reagents

1. When setting upward a PCR experiment, it is of import to be prepared. Habiliment gloves to avoid contaminating the reaction mixture or reagents. Include a negative control, and if possible a positive control.

2. Arrange all reagents needed for the PCR experiment in a freshly filled ice bucket, and allow them thaw completely earlier setting upwardly a reaction (Figure 2). Keep the reagents on water ice throughout the experiment.

   • Standard PCR reagents include a fix of advisable primers for the desired target factor or Deoxyribonucleic acid segment to be amplified, DNA polymerase, a buffer for the specific Dna polymerase, deoxynucleotides (dNTPs), Deoxyribonucleic acid template, and sterile water.

   • Additional reagents may include Magnesium salt Mgⁱⁱ⁺ (at a final concentration of 0.5 to 5.0 mM), Potassium salt K⁺ (at a final concentration of 35 to 100 mM), dimethylsulfoxide (DMSO; at a concluding concentration of one-10%), formamide (at a final concentration of 1.25-x%), bovine serum albumin (at a last concentration of 10-100 μg/ml), and Betaine (at a terminal concentration of 0.5 M to ii.five M). Additives are discussed further in the trouble shooting department.

3. Organize laboratory equipment on the workbench.

   • Materials include PCR tubes and caps, a PCR tube rack, an ethanol-resistant marking, and a set of micropipettors that dispense between ane - 10 μl (P10), 2 - 20 μl (P20), 20 - 200 μl (P200) and 200 - 1000 μl (P1000), as well as a thermal cycler.

   • When setting up several PCR experiments that all use the same reagents, they can exist scaled appropriately and combined together in a main mixture (Master Mix). This pace can be done in a sterile 1.8 ml microcentrifuge tube (encounter Notes).

   • To analyze the amplicons resulting from a PCR experiment, reagents and equipment for agarose gel electrophoresis is required. To approximate the size of a PCR product, an appropriate, commercially bachelor molecular weight size standard is needed.

3. Setting upwards a Reaction Mixture

1. Offset by making a table of reagents that will exist added to the reaction mixture (see Table one).

2. Next, label PCR tube(s) with the ethanol-resistant marker.

3. Reaction volumes will vary depending on the concentrations of the stock reagents. The final concentrations (CF) for a typical 50 μl reaction are equally follows.

   • 10 buffer (commonly supplied past the manufacturer of the DNA polymerase; may comprise 15 mM MgCl₂). Add v μl of 10X buffer per reaction.

   • 200 μM dNTPs (50 μM of each of the four nucleotides). Add 1 μl of ten mM dNTPs per reaction (dATP, dCTP, dTTP and dGTP are at 2.5 mM each).

   • ane.five mM Mg²⁺. Add only if it is not present in the 10X buffer or every bit needed for PCR optimization. For case, to obtain the 4.0 mM Mg²⁺ required for optimal amplicon production of a conserved 566 bp Dna segment found in an uncharacterized Mycobacteriophage add together 8 μl of 25 mM MgCl₂ to the reaction (Figure 3).

   • twenty to 50 pmol of each primer. Add 1 μl of each 20 μM primer.

   • Add ten^iv to 10^vii molecules (or about 1 to 1000 ng) Deoxyribonucleic acid template. Add 0.5 μl of 2ng/μl genomic Mycobacteriophage Dna.

   • Add 0.5 to 2.five units of DNA polymerase per 50 μl reaction (Come across manufacturers recommendations) For example, add 0.v μl of Sigma 0.5 Units/μl Taq DNA polymerase.

   • Add Q.Southward. sterile distilled water to obtain a 50 μl final volume per reaction as pre-determined in the table of reagents (Q.S. is a Latin abbreviation for quantum satis meaning the amount that is needed). Thus, 33 μl per reaction is required to bring the volume up to 50 μl. However, it should exist noted that water is added outset but requires initially making a table of reagents and determining the volumes of all other reagents added to the reaction.

four. Bones PCR Protocol

1. Identify a 96 well plate into the ice saucepan as a holder for the 0.2 ml sparse walled PCR tubes. Allowing PCR reagents to be added into common cold 0.2 ml sparse walled PCR tubes will help prevent nuclease activity and nonspecific priming.

2. Pipette the post-obit PCR reagents in the post-obit order into a 0.ii ml sparse walled PCR tube (Figure four): Sterile H2o, 10X PCR buffer, dNTPs, MgCl_ii, primers, and template DNA (See Table i). Since experiments should accept at least a negative control, and possibly a positive control, information technology is beneficial to gear up a Primary Mix in a 1.8 ml microcentrifuge tube (Meet caption in Notes).

3. In a separate 0.2 ml sparse walled PCR tubes (Effigy 4) add all the reagents with the exception of template Deoxyribonucleic acid for a negative control (increment the water to recoup for the missing volume). In addition, another reaction (if reagents are bachelor) should contain a positive control using template DNA and or primers previously known to amplify nether the same weather as the experimental PCR tubes.

4. Taq Dna polymerase is typically stored in a 50% glycerol solution and for consummate dispersal in the reaction mix requires gentle mixing of the PCR reagents by pipetting up and down at least 20 times. The micropipettor should exist set to nearly half the reaction book of the chief mix when mixing, and intendance should exist taken to avoid introducing bubbles.

5. Put caps on the 0.2 ml thin walled PCR tubes and identify them into the thermal cycler (Effigy 5). Once the lid to the thermal cycler is firmly closed start the program (encounter Table ii).

6. When the program has finished, the 0.2 ml thin walled PCR tubes may be removed and stored at 4 °C. PCR products can be detected by loading aliquots of each reaction into wells of an agarose gel and then staining DNA that has migrated into the gel following electrophoresis with ethidium bromide. If a PCR product is present, the ethidium bromide will intercalate between the bases of the DNA strands, allowing bands to be visualized with a UV illuminator.

Notes:

1. When setting upward multiple PCR experiments, it is advantageous to assemble a mixture of reagents mutual to all reactions (i.e., Main Mix). Commonly the cocktail contains a solution of DNA polymerase, dNTPs, reaction buffer, and h2o assembled into a 1.8 ml microcentrifuge tube. The amount of each reagent added to the Master Mix is equivalent to the full number of reactions plus x% rounded up to the nearest whole reaction. For instance, if there are 10 x 0.1 = 1 reaction, then (10 + i) x 5 μl 10X buffer equals 55 μl of 10X buffer for the Principal Mix. The reagents in the Master Mix are mixed thoroughly by gently pumping the plunger of a micropipettor up and down near 20 times as described above. Each PCR tube receives an aliquot of the Chief Mix to which the DNA template, any required primers, and experiment-specific reagents are then added (come across Tables i and seven).

2. The following website offers a calculator for determining the number of copies of a template DNA (http://www.uri.edu/research/gsc/resource/cndna.html). The total number of copies of double stranded DNA may be calculated using the post-obit equation: Number of copies of Dna = (Deoxyribonucleic acid amount (ng) x 6.022x10²³) / (length of DNA x 1x10⁹ ng/ml x 650 Daltons) Calculating the number of copies of Dna is used to make up one's mind how much template is needed per reaction.

3. False positives may occur every bit a consequence of carry-over from another PCR reaction which would be visualized equally multiple undesired products on an agarose gel later electrophoresis. Therefore, information technology is prudent to use proper technique, include a negative command (and positive command when possible).

4. While ethidium bromide is the about common stain for nucleic acids there are several safer and less toxic alternatives. The following website describes several of the alternatives including Methylene Blue, Crystal Violet, SYBR Safe, and Gel Red along with descriptions of how to employ and detect the last product (http://bitesizebio.com/articles/ethidium-bromide-the-alternatives/).

5. While most modern PCR machines use 0.2 ml tubes, some models may require reactions in 0.v ml tubes. See your thermal cyclers manual to make up one's mind the appropriate size tube.

vi. Setting Up Thermal Cycling Atmospheric condition

1. PCR thermal cyclers rapidly rut and cool the reaction mixture, allowing for heat-induced denaturation of duplex DNA (strand separation), annealing of primers to the plus and minus strands of the Dna template, and elongation of the PCR product. Cycling times are calculated based on the size of the template and the GC content of the DNA. The general formula starts with an initial denaturation pace at 94 °C to 98 °C depending on the optimal temperature for DNA polymerase activity and G-C content of the template DNA. A typical reaction will commencement with a one minute denaturation at 94 °C. Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. One method, known as hot-offset PCR, drastically extends the initial denaturation time from three minutes upwards to 9 minutes. With hot-start PCR, the Dna polymerase is added after the initial exaggerated denaturation stride is finished. This protocol modification avoids likely inactivation of the DNA polymerase enzyme. Refer to the Troubleshooting section of this protocol for more than data nearly hot start PCR and other alternative methods.

2. The next stride is to set the thermal cycler to initiate the beginning of 25 to 35 rounds of a three-stride temperature cycle (Tabular array ii). While increasing the number of cycles above 35 will effect in a greater quantity of PCR products, as well many rounds often results in the enrichment of undesirable secondary products. The iii temperature steps in a single cycle attain iii tasks: the first step denatures the template (and in afterward cycles, the amplicons as well), the 2d footstep allows optimal annealing of primers, and the third stride permits the Deoxyribonucleic acid polymerase to bind to the DNA template and synthesize the PCR product. The elapsing and temperature of each step within a wheel may be contradistinct to optimize production of the desired amplicon. The time for the denaturation pace is kept as short as possible. Usually x to 60 seconds is sufficient for nigh Deoxyribonucleic acid templates. The denaturation time and temperature may vary depending on the G-C content of the template DNA, as well equally the ramp charge per unit, which is the time it takes the thermal cycler to change from ane temperature to the next. The temperature for this stride is usually the same as that used for the initial denaturation phase (footstep #1 to a higher place; e.g., 94 °C). A 30 second annealing step follows within the bicycle at a temperature set near 5 °C beneath the credible T_g of the primers (ideally betwixt 52 °C to 58 °C). The bicycle concludes with an elongation step. The temperature depends on the DNA polymerase selected for the experiment. For example, Taq Deoxyribonucleic acid polymerase has an optimal elongation temperature of 70 °C to 80 °C and requires one minute to elongate the first two kb, so requires an extra infinitesimal for each boosted i kb amplified. Pfu DNA Polymerase is some other thermostable enzyme that has an optimal elongation temperature of 75 °C. Pfu DNA Polymerase is recommended for utilize in PCR and primer extension reactions that require high fidelity and requires ii minutes for every 1 kb to be amplified. See manufacturer recommendations for exact elongation temperatures and elongation fourth dimension indicated for each specific Deoxyribonucleic acid polymerase.

3. The concluding phase of thermal cycling incorporates an extended elongation menstruation of 5 minutes or longer. This last step allows synthesis of many uncompleted amplicons to terminate and, in the case of Taq DNA polymerase, permits the add-on of an adenine residual to the 3' ends of all PCR products. This modification is mediated by the terminal transferase action of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that require a iii'-overhang.

4. Termination of the reaction is accomplished by chilling the mixture to four °C and/or past the addition of EDTA to a concluding concentration of 10 mM.

vii. Of import Considerations When Troubleshooting PCR

If standard PCR conditions do not yield the desired amplicon, PCR optimization is necessary to attain better results. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables (e.g., reagent concentrations, cycling atmospheric condition) that affect the consequence of the amplicon profile. For case, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is besides stringent, no product will be produced. Troubleshooting PCR reactions may be a frustrating endeavor at times. However, conscientious analysis and a good understanding of the reagents used in a PCR experiment tin reduce the corporeality of time and trials needed to obtain the desired results. Of all the considerations that impact PCR stringency, titration of Mgⁱⁱ⁺ and/or manipulating annealing temperatures likely will solve virtually issues. Yet, before changing annihilation, exist sure that an erroneous result was not due to human error. First by confirming all reagents were added to a given reaction and that the reagents were not contaminated. Also take note of the erroneous consequence, and ask the following questions: Are primer dimers visible on the gel after electrophoresis (these run equally small bands <100 b near the bottom of the lane)? Are there non-specific products (bands that drift at a different size than the desired production)? Was there a lack of any product? Is the target DNA on a plasmid or in a genomic DNA extract? Also, information technology is wise to analyze the G-C content of the desired amplicon.

1. First determine if any of the PCR reagents are catastrophic to your reaction. This can be achieved by preparing new reagents (east.thou., fresh working stocks, new dilutions), and then systematically adding one new reagent at a fourth dimension to reaction mixtures. This process will determine which reagent was the culprit for the failed PCR experiment. In the instance of very old Dna, which oft accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may assistance alleviate the trouble.

2. Primer dimers tin course when primers preferentially self amalgamate or anneal to the other primer in the reaction. If this occurs, a small product of less than 100 bp will announced on the agarose gel. Kickoff past altering the ratio of template to primer; if the primer concentration is in extreme excess over the template concentration, then the primers volition be more than probable to anneal to themselves or each other over the DNA template. Adding DMSO and or using a hot commencement thermal cycling method may resolve the problem. In the finish it may be necessary to design new primers.

3. Non-specific products are produced when PCR stringency is excessively low resulting in non-specific PCR bands with variable lengths. This produces a ladder effect on an agarose gel. It then is advisable to choose PCR conditions that increase stringency. A smear of various sizes may likewise result from primers designed to highly repetitive sequences when amplifying genomic DNA. Nonetheless, the aforementioned primers may amplify a target sequence on a plasmid without encountering the aforementioned problem.

4. Lack of PCR products is probable due to reaction conditions that are likewise stringent. Primer dimers and hairpin loop structures that course with the primers or in the denatured template Deoxyribonucleic acid may as well prevent amplification of PCR products because these molecules may no longer base pair with the desired DNA analogue.

5. If the G-C content has not been analyzed, it is fourth dimension to practise so. PCR of Thousand-C rich regions (GC content >60%) pose some of the greatest challenges to PCR. Even so, at that place are many additives that have been used to help alleviate the challenges.

8. Manipulating PCR Reagents

Understanding the function of reagents used on conventional PCR is critical when outset deciding how best to alter reaction conditions to obtain the desired production. Success simply may rely on changing the concentration of MgCl₂, KCl, dNTPs, primers, template Deoxyribonucleic acid, or DNA polymerase. However, the wrong concentration of such reagents may lead to spurious results, decreasing the stringency of the reaction. When troubleshooting PCR, only 1 reagent should be manipulated at a time. Yet, information technology may exist prudent to titrate the manipulated reagent.

1. Magnesium salt Mg^two+ (final reaction concentration of 0.5 to 5.0 mM) Thermostable Dna polymerases require the presence of magnesium to act as a cofactor during the reaction process. Changing the magnesium concentration is i of the easiest reagents to manipulate with perhaps the greatest impact on the stringency of PCR. In general, the PCR product yield volition increment with the addition of greater concentrations of Mgⁱⁱ⁺. Yet, increased concentrations of Mgⁱⁱ⁺ will likewise decrease the specificity and fidelity of the DNA polymerase. Most manufacturers include a solution of Magnesium chloride (MgCl₂) along with the Dna polymerase and a 10X PCR buffer solution. The 10 X PCR buffer solutions may contain fifteen mM MgCl₂, which is plenty for a typical PCR reaction, or information technology may be added separately at a concentration optimized for a particular reaction. Mg²⁺ is non actually consumed in the reaction, but the reaction cannot continue without it being nowadays. When in that location is too much Mg²⁺, it may prevent complete denaturation of the Deoxyribonucleic acid template past stabilizing the duplex strand. As well much Mg^two+ too tin stabilize spurious annealing of primers to incorrect template sites and decrease specificity resulting in undesired PCR products. When there is not plenty Mg²⁺, the reaction will non proceed, resulting in no PCR production.

2. Potassium table salt One thousand⁺ (final reaction concentration of 35 to 100 mM) Longer PCR products (10 to forty kb) benefit from reducing potassium salt (KCl) from its normal 50 mM reaction concentration, often in conjunction with the addition of DMSO and/or glycerol. If the desired amplicon is below grand bp and long not-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to 100 mM. Increasing the common salt concentration permits shorter Dna molecules to denature preferentially to longer Dna molecules.

3. Deoxynucleotide 5'-triphosphates (final reaction concentration of 20 and 200 μM each) Deoxynucleotide v'-triphosphates (dNTPs) can cause problems for PCR if they are not at the appropriate equivalent concentrations (i.eastward., [A] = [T] = [C] = [K]) and/ or due to their instability from repeated freezing and thawing. The usual dNTP concentration is 50 μM of EACH of the four dNTPs. However, PCR can tolerate concentrations between 20 and 200 μM each. Lower concentrations of dNTPs may increase both the specificity and fidelity of the reaction while excessive dNTP concentrations tin can really inhibit PCR. However, for longer PCR-fragments, a college dNTP concentration may exist required. A large modify in the dNTP concentration may necessitate a corresponding alter in the concentration of Mg²⁺.

4. Thermal stable Dna polymerases PCR enzymes and buffers associated with those enzymes have come a long way since the initial Taq DNA polymerase was get-go employed. Thus, choosing an appropriate enzyme can exist helpful for obtaining desired amplicon products. For example the use of Taq DNA polymerase may be preferred over Pfu Deoxyribonucleic acid polymerase if processivity and/or the add-on of an adenine rest to the 3' ends of the PCR production is desired. The add-on of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. However, if fidelity is more important an enzyme such as Pfu may be a better choice. Several manufactures have an assortment of specific DNA polymerases designed for specialized needs. Take a look at the reaction atmospheric condition and characteristics of the desired amplicon, and so lucifer the PCR experiment with the appropriate DNA polymerase. About manufactures have tables that aid Dna polymerase selection past list characteristics such as allegiance, yield, speed, optimal target lengths, and whether information technology is useful for One thousand-C rich amplification or hot start PCR.

5. Template DNA Deoxyribonucleic acid quality and purity will accept a substantial effect on the likelihood of a successful PCR experiment. DNA and RNA concentrations can be determined using their optical density measurements at 260 nm (OD₂₆₀). The mass of purified nucleic acids in solution is calculated at l μg/ml of double stranded DNA or xl μg/ml for either RNA or unmarried stranded DNA at an OD₂₆₀ =1.0. DNA extraction contaminants are common inhibitors in PCR and should be carefully avoided. Common Deoxyribonucleic acid extraction inhibitors of PCR include protein, RNA, organic solvents, and detergents. Using the maximum absorption of nucleic acids OD₂₆₀ compared to that of proteins OD280 (OD_260/280), it is possible to determine an estimate of the purity of extracted DNA. Ideally, the ratio of OD_260/280 is betwixt 1.8 and 2.0. Lower OD_260/280 is indicative of protein and/ or solvent contamination which, in all probability, volition be problematic for PCR. In improver to the quality of template Deoxyribonucleic acid, optimization of the quantity of Deoxyribonucleic acid may greatly benefit the outcome of a PCR experiment. Although it is convenient to determine the quantity in ng/μl, which is often the output for modern nanospectrophotometers, the relevant unit for a successful PCR experiment is the number of molecules. That is, how many copies of Deoxyribonucleic acid template contain a sequence complementary to the PCR primers? Optimal target molecules are between ten^iv to 10⁷ molecules and may be calculated as was described in the notes to a higher place.

9. Additive Reagents

Additive reagents may yield results when all else fails. Agreement the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product. Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may affect the usable concentration of another reagent. In addition to the reagents listed beneath, proprietary commercially bachelor additives are available from many biotechnology companies.

10. Additives That Benefit G-C Rich Templates

1. Dimethylsulfoxide (concluding reaction concentration of 1-10% DMSO) In PCR experiments in which the template DNA is especially G-C rich (GC content >lx%), calculation DMSO may enhance the reaction by disrupting base pairing and effectively lowering the T_yard. Some T_chiliad calculators include a variable entry for adding the concentration of DMSO desired in the PCR experiment. All the same, adding more than two% DMSO may require adding more Dna polymerase as it has been demonstrated to inhibit Taq DNA polymerase.

2. Formamide (final reaction concentration of 1.25-ten%) Like DMSO, formamide also disrupts base pairing while increasing the stringency of primer annealing, which results in less non-specific priming and increased amplification efficiency. Formamide likewise has been shown to be an enhancer for G-C rich templates.

3. vii-deaza-2'-deoxyguanosine 5'-triphosphate (final reaction concentration of dc⁷GTP; iii dc^viiGTP:ane dGTP fifty μM) Using 3 parts, or 37.five μM, of the guanosine base of operations analog dc^viiGTP in conjunction with 1 role, or 12.v μM, dGTP will destabilize formation of secondary structures in the product. Equally the amplicon or template DNA is denatured, information technology will oft form secondary structures such every bit hairpin loops. Incorporation of dc⁷GTP into the DNA amplicon volition prohibit formation of these aberrant structures.

Note:

dc⁷GTP attenuates the point of ethidium bromide staining which is why it is used in a 3:1 ratio with dGTP.

1. Betaine (final reaction concentration of 0.5M to 2.5M) Betaine (North,N,N-trimethylglycine) is a zwitterionic amino acid analog that reduces and may even eliminate the Dna melting temperature dependence on nucleotide composition. Information technology is used as an condiment to assist PCR amplification of Grand-C rich targets. Betaine is oft employed in combination with DMSO and can greatly raise the chances of amplifying target DNA with high Grand-C content.

11. Additives That Aid PCR in the Presence of Inhibitors

1. Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase. Non ionic detergents such every bit Triton 10-100, Tween 20, or NP-xl may be used at reaction concentrations of 0.ane to 1% in guild to increase amplicon production. Nevertheless, concentrations higher up ane% may exist inhibitory to PCR. The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious production formation. However, their apply will likewise neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols.

2. Addition of specific proteins such as Bovine serum albumin (BSA) used at 400 ng/μl and/ or T4 gene 32 protein at 150 ng/μl aid PCR in the presence of inhibitors such as FeCl₃, hemin, fulvic acid, humic acid, tannic acids, or extracts from carrion, fresh h2o, and marine water. However, some PCR inhibitors, including bile salts, bilirubin, EDTA, NaCl, SDS, or Triton X-100, are not alleviated by addition of either BSA or T4 gene 32 protein.

12. Modifications to Cycling Weather condition

1. Optimizing the annealing temperature volition heighten whatever PCR reaction and should be considered in combination with other additives and/ or along with other modifications to cycling atmospheric condition. Thus, in guild to calculate the optimal annealing temperature the following equation is employed: T_a ^OPT = 0.3 T_thou ^Primer + 0.7 T_thou ^Product -14.9 T₁₀₀₀ ^Primer is calculated as the T_m of the less stable pair using the equation: T_yard ^Primer = ((ΔH/(ΔS+R 10 ln(c/4)))-273.xv + 16.6 log[K⁺] Where ΔH is the sum of the nearest neighbor enthalpy changes for hybrids; ΔS is the sum of the nearest neighbor entropy changes; R is the Gas Constant (1.99 cal 1000-1 mol-1); C is the primer concentration; and [G⁺] is the potassium concentration. The latter equation can be computed using 1 of the T_g calculators listed at the post-obit website: http://protein.bio.puc.cl/cardex/servers/melting/sup_mat/servers_list.html T_m ^Product is calculated as follows: T _thou ^Product = 0.41(%One thousand-C) + 16.half dozen log [K⁺] - 675/product length For most PCR reactions the concentration of potassium ([Chiliad⁺]) is going to be l mM.

2. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically (Table 4). This modification can be incorporated with or without other modifications to cycling atmospheric condition. Moreover, information technology is often used in conjunction with additives for temperamental amplicon germination. In fact, hot start PCR is increasingly included equally a regular aspect of general cycling conditions. Hot starting time has been demonstrated to increment amplicon yield, while increasing the specificity and fidelity of the reaction. The rationale backside hot starting time PCR is to eliminate primer-dimer and non-specific priming that may result as a upshot of setting up the reaction below the T_m. Thus, a typical hot start reaction heats the sample to a temperature higher up the optimal T_chiliad, at least to 60 °C before any amplification is able to occur. In general, the Deoxyribonucleic acid polymerase is withheld from the reaction during the initial, elongated, denaturing time. Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase. At that place are several methods which allow the Dna polymerase to remain inactive or physically separated until the initial denaturation menstruum has completed, including the use of a solid wax bulwark, anti-Deoxyribonucleic acid polymerase antibodies, and accessory proteins. Alternatively, the DNA polymerase may but exist added to the reaction after the initial denaturation cycle is consummate.

3. Touchdown PCR (TD-PCR) is intended to take some of the judge work out of the T_{one thousand} calculation limitations by bracketing the calculated annealing temperatures. The concept is to pattern two phases of cycling conditions (Tabular array 5). The starting time stage employs successively lower annealing temperatures every second bicycle (traditionally i.0 °C), starting at x °C above and finishing at the calculated T_m or slightly below. Stage two utilizes the standard 3-step weather with the annealing temperature gear up at 5 °C below the calculated T_m for some other 20 to 25 cycles. The function of the showtime phase should convalesce mispriming, conferring a 4-fold advantage to the correct product. Thus, subsequently 10 cycles, a 410-fold reward would yield 4096 copies of the correct product over whatsoever spurious priming.

   • Stepdown PCR is similar to TD-PCR with fewer increments in the beginning phase of priming. As an case, the first phase lowers annealing temperatures every second wheel by 3 °C, starting at ten °C above and finishing at ii °C below the calculated T_m. Like TD-PCR, stage two utilizes the standard 3-stride conditions with the annealing temperature set at v °C below the calculated T_g for another 20 to 25 cycles. This would let the correct product a 256-fold reward over false priming products.

   • Slowdown PCR is just a modification of TD-PCR and has been successful for amplifying extremely G-C rich (above 83%) sequences (Tabular array six). The concept takes into account a relatively new characteristic associated with modern thermal cyclers, which allows adjustment of the ramp speed as well as the cooling rate. The protocol also utilizes dc^sevenGTP to reduce ii °structure formation that could inhibit the reaction. The ramp speed is lowered to two.v °C s^-1 with a cooling rate of 1.v °C s^-1 for the annealing cycles. The beginning phase starts with an annealing temperature of 70 °C and reduces the annealing temperature by 1 °C every 3 rounds until information technology reaches 58 °C. The second phase and then continues with an annealing temperature of 58 °C for an additional 15 cycles.

4. Nested PCR is a powerful tool used to eliminate spurious products. The use of nested primers is particularly helpful when there are several paralogous genes in a unmarried genome or when in that location is low copy number of a target sequence inside a heterogeneous population of orthologous sequences. The basic process involves 2 sets of primers that dilate a unmarried region of DNA. The outer primers straddle the segment of interest and are used to generate PCR products that are ofttimes not-specific in 20 to 30 cycles. A minor aliquot, usually about v μl from the starting time l μl reaction, is and so used as the template DNA for another 20 to 30 rounds of amplification using the second set of primers that amalgamate to an internal location relative to the first gear up.

Other PCR protocols are more specialized and go beyond the scope of this paper. Examples include RACE-PCR, Multiplex-PCR, Vectorette-PCR, Quantitative-PCR, and RT-PCR.

13. Representative Results

Representative PCR results were generated by following the basic PCR protocols described above. The results contain several troubleshooting strategies to demonstrate the effect of various reagents and conditions on the reaction. Genes from the budding yeast Saccharomyces cerevisiae and from an uncharacterized Mycobacteriophage were amplified in these experiments. The standard iii-step PCR protocol outlined in Tabular array 2 was employed for all three experiments described below.

Before setting upwards the PCR experiment, the genomic DNA from both South. cerevisiae and the Mycobacteriophage were quantified and diluted to a concentration that would permit between x⁴ and 10⁷ molecules of DNA per reaction. The working stocks were prepared as follows. A genomic yeast DNA preparation yielded 10^iv ng/μl. A dilution to 10 ng/μl was generated past adding 48 μl into 452 μl of TE pH 8.0 buffer. Since the S. cerevisiae genome is well-nigh 12.5 Mb, x ng contain 7.41 X ten⁵ molecules. The genomic Mycobacteriophage DNA training yielded 313 ng/μl. A dilution to 2 ng/μl was generated past adding vi.4 μl into 993.6 μl of TE pH 8.0 buffer. This phage Deoxyribonucleic acid is about 67 Kb. Thus, 1 ng contains 2.73 X 10⁷ molecules, which is at the upper limit of DNA generally used for a PCR. The working stocks were then used to generate the Master Mix solutions outlined in Table 7. Experiments varied cycling conditions equally described beneath.

In Figure 3a, genomic DNA from S. cerevisiae was used as a template to amplify the GAL3 cistron, which encodes a protein involved in galactose metabolism. The goal for this experiment was to determine the optimal Mg²⁺ concentration for this prepare of reagents. No MgCl₂ was present in the original PCR buffer and had to be supplemented at the concentrations indicated with a range tested from 0.0 mM to 5.0 mM. As shown in the effigy, a PCR production of the expected size (2098 bp) appears starting at a Mgⁱⁱ⁺ concentration of 2.five mM (lane six) with an optimal concentration at iv.0 mM (lane nine). The recommended concentration provided by the manufacturer was ane.5 mM, which is the amount provided in typical PCR buffers. Mayhap surprisingly, the necessary concentration needed for product germination in this experiment exceeded this corporeality.

A dissimilar Dna template was used for the experiment presented in Figure 3b. Genomic DNA from a Mycobacteriophage was used to dilate a conserved 566 bp DNA segment. Like the previous experiment, the optimal Mg²⁺ concentration had to be determined. As shown in Figure 3b, amplification of the desired PCR production requires at least 2.0 mM Mg^two+ (lane five). While at that place was more variability in the amount of product formed at increasing concentrations of MgCl₂, the about PCR production was observed at 4 mM Mgⁱⁱ⁺ (lane nine), the same concentration observed for the yeast GAL3 cistron.

Notice that in the experiments presented in Figures 3A and 3B, a discrete band was obtained using the cycling conditions thought to be optimal based on primer annealing temperatures. Specifically, the denaturation temperature was 95 °C with an annealing temperature of 61 °C, and the extension was carried out for 1 infinitesimal at 72 °C for xxx cycles. The final 5 infinitesimal extension was then done at 72 °C. For the third experiment presented in Effigy 3c, three changes were made to the cycling conditions used to amplify the yeast GAL3 gene. Commencement, the annealing temperature was reduced to a sub-optimal temperature of 58 °C. Second, the extension time was extended to 1 minute and 30 seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal amplification weather condition (i.e., reducing the stringency of the reaction) on a PCR experiment. As shown in Figure 3c, what was a discrete ring in Figure 3a, becomes a smear of non-specific products under these sub-optimal cycling conditions. Furthermore, with the overall stringency of the reaction reduced, a lower amount of Mgⁱⁱ⁺ is required to form an amplicon.

All iii experiments illustrate that when Mg²⁺ concentrations are too depression, in that location is no amplicon product. These results also demonstrate that when both the cycling conditions are correctly designed and the reagents are at optimal concentrations, the PCR experiment produces a unimposing amplicon corresponding to the expected size. The results bear witness the importance of performing PCR experiments at a sufficiently high stringency (eastward.g., discreet bands versus a smear). Moreover, the experiments bespeak that changing one parameter tin influence another parameter, thus affecting the reaction effect.

Reagent	Concentration of stock solutions	Book	13X ** Master Mix	Final Concentration
Sterile HiiO		Q.S. to 50 μl	Q.S. to 650 μl
PCR Buffer	10X	5 μl	65 μl	1X
dNTP'due south	10 mM	ane μl	xiii μl	200 μM
MgClii	25 mM	iii μl	39 μl	one.5 mM
Frontward Primer	20 μM = 20 pmol/μl	one μl	13 μl	20 pmol
Reverse Primer	20 μM = 20 pmol/μl	ane μl	13 μl	twenty pmol
Template DNA	Variable	Variable	Variable	~xfive Molecules
Taq Dna Polymerase	v Units/μl*	0.5 μl	six.5 μl	2.five Units
				50 μl/Reaction

Table i. PCR reagents in the order they should exist added.

*Units may vary between manufacturers

** Add all reagents to the Main Mix excluding any in demand of titration or that may be variable to the reaction. The Primary Mix depicted in the above table is calculated for 11 reactions plus ii extra reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube.

	Standard 3-footstep PCR Cycling
Cycle pace	Temperature	Time	Number of Cycles
Initial Denaturation	94 °C to 98 °C	1 minute	1
Denaturation Annealing Extension	94 °C 5 °C below Tk lxx °C to lxxx °C	ten to 60 seconds 30 seconds Amplicon and DNA polymerase dependent	25-35
Final Extension	lxx °C to fourscore °C	5 minutes	1
Hold*	4 °C	∞	1

Tabular array 2. Standard 3-step PCR Cycling.

* About thermal cyclers have the ability to pause at 4°C indefinitely at the terminate of the cycles.

	two-step PCR Cycling
Cycle step	Temperature	Time	Number of Cycles
Initial Denaturation	94 °C to 98 °C	1 minute	1
Denaturation Annealing/Extension	94 °C 70 °C to 80 °C	ten to 60 seconds Amplicon and Dna polymerase dependent	25-35
Last Extension	70 °C to lxxx °C	v minutes	1

Table 3. two-footstep PCR Cycling.

	Hot Outset PCR Cycling
Cycle stride	Temperature	Time	Cycles
Initial Denaturation	60 °C to 95 °C	5 minute then add DNA polymerase	1
Denaturation Annealing Extension	94 °C five °C below Tyard 70 °C to lxxx °C	10 to 60 seconds 30 seconds Amplicon and DNA polymerase dependent	25-35
Concluding Extension	70 °C to eighty °C	5 minutes	1

Table 4. Hot Start PCR Cycling.

	Touchdown PCR Cycling
Bicycle step	Temperature	Time	Cycles
Initial Denaturation	94 °C to 98 °C	1 minute	1
Denaturation Annealing Extension	94 °C X =10 °C in a higher place Tm 70 °C to fourscore °C	x to 60 seconds thirty seconds Amplicon and DNA polymerase dependent	2
Denaturation Annealing Extension	94 °C X-one °C reduce i °C every other cycle seventy °C to lxxx °C	ten to 60 seconds xxx seconds Amplicon and polymerase dependent	28
Denaturation Annealing Extension	94 °C five °C beneath Tm 70 °C to 80 °C	10 to 60 seconds 30 seconds Amplicon and DNA polymerase dependent	20-25
Final Extension	70 °C to 80 °C	v minutes	1

Table v. Touchdown PCR Cycling.

	Slowdown PCR Cycling
Cycle step	Temperature	Time	Cycles
Initial Denaturation	94 °C to 98 °C	i minute	1
Denaturation Annealing Extension	94 °C Ten °C =x °C above T1000 seventy °C to 80 °C	ten to 60 seconds 30 seconds Amplicon and polymerase dependent	2
Denaturation Annealing Extension	94 °C X-one °C reduce 1 °C every other wheel 70 °C to lxxx °C*	x to 60 seconds xxx seconds Amplicon and polymerase dependent	28
Denaturation Annealing Extension	94 °C 5 °C beneath Tchiliad 70 °C to 80 °C	10 to 60 seconds xxx seconds Amplicon and polymerase dependent	xx-25
Concluding Extension	70 °C to 80 °C	5 minutes	ane

Table six. Slowdown PCR Cycling.

*For slowdown PCR, the ramp speed is lowered to 2.5 °C south^-1 with a cooling rate of 1.5 °C s^-1 for the annealing cycles.

	Stock Solution	Book added to 50 μl reaction	xiii X Yeast Master Mix	13 X Phage Principal Mix	Final Concentration
Sterile HiiO	q.south. to fifty μl = 31 μl or 30.5	q.due south. to 650 μl = 396.5	q.south. to 520 μl = 403 μl
PCR Buffer	10X	v μl	65 μl	65 μl	1X
dNTP's	10 mM	1 μl	13 μl	13 μl	200 μM
MgCl2	Titration	Added to each reaction	Added to each reaction	Added to each reaction	Variable encounter titration
Forward Primer	twenty μM = 20 pmol/μl	1 μl	13 μl	13 μl	twenty pmol
Reverse Primer	20 μM = 20 pmol/μl	1 μl	13 μl	13 μl	twenty pmol
Template DNA	ii ng/μl phage or 10 ng/μl Yeast	0.five μl Phage or 1 μl Yeast	6.five μl	13 μl	~x7 Molecules Phage or ~10v Molecules Yeast
Polymerase	0.5 Units/μl**	0.5 μl	6.v μl	vi.five μl	0.5 Units/Reaction
					40 μl + x(Titration) μl/ Reaction
TITRATION
[MgCl2]	0.00 mM	0.5 mM	1.0 mM	1.5mM	2.0 mM	2.5 mM	iii.0 mM	iii.5 mM	iv.0 mM	4.5 mM	5.0 mM
MgCl2	0.00 μl	i.00 μl	2.00 μl	3.0 μl	4.00 μl	5.00 μl	6.00 μl	vii.00 μl	8.00 μl	9.00 μl	10.00 μl
H2O	ten.00 μl	9.00 μl	8.00 μl	7.00 μl	6.00 μl	5.00 μl	iv.00 μl	3.00 μl	2.00 μl	i.00 μl	0.00 μl

Tabular array 7. Titration of Mg²⁺ used in Figure 3.

Figure one. Common problems that ascend with primers and 3-pace PCR amplification of target DNA. (a) Cocky-annealing of primers resulting in formation of secondary hairpin loop structure. Note that primers do not e'er amalgamate at the farthermost ends and may form smaller loop structures. (b) Primer annealing to each other, rather than the Deoxyribonucleic acid template, creating primer dimers. Once the primers amalgamate to each other they will elongate to the primer ends. (c) PCR cycles generating a specific amplicon. Standard 3-step PCR cycling include denaturation of the template Dna, annealing of primers, and extension of the target DNA (amplicon) by DNA polymerase.

Figure 2. Ice bucket with reagents, pipettes, and racks required for a PCR. (1.) P-200 pipette, (2.) P-1000 pipette, (3.) P-20 pipette, (four.) P-10 pipette, (5.) 96 well plate and 0.ii ml sparse walled PCR tubes, (six.) Reagents including Taq polymerase, 10X PCR buffer, MgCl_ii, sterile water, dNTPs, primers, and template Deoxyribonucleic acid, (vii.) 1.viii ml tubes and rack.

Figure iii. Case of a Mg^two+ titrations used to optimize a PCR experiment using a standard 3-step PCR protocol. (a) Due south. cerevisiae Yeast genomic DNA was used as a template to dilate a 2098 bp GAL3 factor. In lanes 1 - half-dozen, where the Mg²⁺ concentration is too depression, there either is no product formed (lanes 1-5) or very little product formed (lane 6). Lanes vii - 11 stand for optimal concentrations of Mg²⁺ for this PCR experiment as indicated by the presence of the 2098 bp amplicon production. (b) An uncharacterized mycobacteriophage genomic Deoxyribonucleic acid template was used to dilate a 566 bp amplicon. Lanes one - iv, the Mg^two+ concentration is too low, as indicated by the absenteeism of production. Lanes 5 - 11 represent optimal concentrations of Mgⁱⁱ⁺ for this PCR as indicated past the presence of the 566 kb amplicon product. (c) . Southward. cerevisiae Yeast genomic DNA was used as a template to amplify a 2098 bp GAL3 cistron equally indicated in panel a. However, the annealing temperature was reduced from 61 °C to 58 °C, resulting in a non-specific PCR bands with variable lengths producing a smearing effect on the agarose gel. Lanes 1 - four, where the Mgⁱⁱ⁺ concentration is too low, there is no product formed. Lanes 5 - 8 represent optimal concentrations of Mgⁱⁱ⁺ for this PCR as seen by the presence of a smear and band around the 2098 kb amplicon product size. Lanes 9 - 11 are indicative of excessively stringent conditions with no product formed. (a-c) Lanes 12 is a negative control that did not comprise any template Dna. Lane M (marker) was loaded with NEB 1kb Ladder.

Figure 4. Sterile tubes used for PCR. (ane.) 1.8 ml tube (ii.) 0.2 ml individual thin walled PCR tube, (3.) 0.ii ml strip thin walled PCR tubes and caps.

Figure 5. Thermal cycler. Closed thermal cycler left image. Right paradigm contains 0.2 ml thin walled PCR tubes placed in the heating block of an open up thermal cycler.

Discussion

PCR has become an indispensible tool in the biological science arsenal. PCR has contradistinct the grade of scientific discipline allowing biologists to yield power over genomes, and brand hybrid genes with novel functions, assuasive specific and accurate clinical testing, gaining insights into genomes and variety, as well as only cloning genes for further biochemical analysis. PCR application is express just by the imagination of the scientist that wields its power. There are many books and papers that depict new specialized uses of PCR, and many more will be developed over the next generation of biology. However, regardless of the predictable approaches, the fundamental framework has remained the same. PCR, in all its grandeur, is an in vitro application to generate large quantities of a specific segment of DNA.

Designing a PCR experiment requires thought and patience. The results shown in Effigy three exemplify one of the major challenges when designing an optimization strategy for PCR. That is, as 1 parameter of PCR is inverse, it may bear on another. As an instance, if the initial PCR was carried out at the sub-optimal annealing temperature (58°C) with an optimal Mg^two+ concentration of 2.0 mM, then the event would produce a smear as seen in Figure 3c. An endeavor to resolve the smear might involve setting up PCR conditions with reactions containing 2.0 mM MgCl_two and adjusting the annealing temperature to 61°C. Even so, as seen in Figure 3a, this would not yield whatever production. Consequently, it is advisable to titrate reagents, rather than adding i concentration to a single reaction, when troubleshooting spurious results. Besides, the most common adjustments that are required for optimizing a PCR experiment are to change the Mg²⁺ concentration and to right the annealing temperatures. However, if these changes do not minimize or abrogate abnormal results, titration of additives and /or irresolute the cycling condition protocols as described in Tables 2-6 may alleviate the problem. If all else fails, redesign the primers and try, try once again.

Disclosures

I have nix to disclose.

Acknowledgments

Special thanks to Kris Reddi at UCLA for setting up reagents and pouring gels and to Erin Sanders at UCLA for inspiration, guidance, and support and proofreading the manuscript. I would also like to thank you Giancarlo Costaguta and Gregory South. Payne for supplying the yeast genomic Dna and primers to amplify the GAL3 factor. I would also similar to thank Bhairav Shah for taking pictures of the lab equipment and reagents used to make figures 2 - 4. Funding for this project was provided by HHMI (HHMI Grant No. 52006944).

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