How Do You Know If Your Pcr Amplification Is Clean Enough?

Abstract

In the biological sciences there have been technological advances that catapult the discipline into gilt ages of discovery. For example, the field of microbiology was transformed with the appearance of Anton van Leeuwenhoek'due south microscope, which allowed scientists to visualize prokaryotes for the starting time time. The development of the polymerase concatenation reaction (PCR) is one of those innovations that changed the course of molecular science with its bear upon spanning countless subdisciplines in biology. The theoretical procedure was outlined past Keppe and coworkers in 1971; however, it was another xiv 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 Dna polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.

PCR is a powerful amplification technique that tin can generate an ample supply of a specific segment of Dna (i.e., an amplicon) from only a small corporeality of starting material (i.e., Deoxyribonucleic acid template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can pb to many non-specific DNA products of varying sizes that appear every bit a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another 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(south). This protocol outlines the bones principles of PCR, provides a methodology that will issue in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should exist able to: ● Set upwardly reactions and thermal cycling conditions for a conventional PCR experiment ● Sympathize the office of various reaction components and their overall effect on a PCR experiment ● Design and optimize a PCR experiment for whatsoever Dna template ● Troubleshoot failed PCR experiments

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

Protocol

ane. Designing Primers

Designing appropriate primers is essential to the successful consequence of a PCR experiment. When designing a set of primers to a specific region of Deoxyribonucleic acid desired for amplification, one primer should anneal to the plus strand, which by convention is oriented in the v' → 3' management (also known as the sense or nontemplate strand) and the other primer should complement the minus strand, which is oriented in the 3' → v' direction (antisense or template strand). There are a few common problems that arise when designing primers: 1) self-annealing of primers resulting in germination of secondary structures such as hairpin loops (Figure 1a); ii) primer annealing to each other, rather then the DNA template, creating primer dimers (Figure 1b); 3) drastically different melting temperatures (T_thousand) 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_thou) and MODIFICATIONS TO CYCLING Atmospheric condition for more information on T_yards).

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

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

   2. Optimal G-C content should range betwixt 40-threescore%.

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

   4. The 3' ends of a primer prepare, 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 be complementary to other sequences in the primer. These two scenarios result in formation of primer dimers and hairpin loop structures, respectively.

   5. Optimal melting temperatures (T_thou) for primers range betwixt 52-58 °C, although the range can be expanded to 45-65 °C. The final T_thou for both primers should differ by no more than v °C.

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

Notes:

1. There are many computer programs designed to help in designing primer pairs. NCBI Primer design 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 avoid distension of related pseudogenes or homologs it could be useful to run a blast on NCBI to bank check for the target specificity of the primers.

2. Materials and Reagents

1. When setting up a PCR experiment, information technology is important to be prepared. Wear gloves to avoid contaminating the reaction mixture or reagents. Include a negative command, and if possible a positive control.

2. Arrange all reagents needed for the PCR experiment in a freshly filled ice saucepan, and let them thaw completely before setting up a reaction (Figure two). Keep the reagents on ice throughout the experiment.

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

   • Additional reagents may include Magnesium salt Mg²⁺ (at a final concentration of 0.5 to 5.0 mM), Potassium common salt K⁺ (at a final concentration of 35 to 100 mM), dimethylsulfoxide (DMSO; at a final concentration of ane-10%), formamide (at a terminal concentration of i.25-10%), bovine serum albumin (at a terminal concentration of 10-100 μg/ml), and Betaine (at a final concentration of 0.5 K to 2.five M). Additives are discussed farther in the trouble shooting section.

3. Organize laboratory equipment on the workbench.

   • Materials include PCR tubes and caps, a PCR tube rack, an ethanol-resistant marker, and a set up of micropipettors that manipulate betwixt one - 10 μl (P10), 2 - twenty μl (P20), 20 - 200 μl (P200) and 200 - thousand μl (P1000), every bit well as a thermal cycler.

   • When setting up several PCR experiments that all use the aforementioned reagents, they can exist scaled appropriately and combined together in a chief mixture (Principal Mix). This pace tin be done in a sterile 1.8 ml microcentrifuge tube (see 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.

iii. Setting up a Reaction Mixture

1. Start past making a table of reagents that will be added to the reaction mixture (encounter Table ane).

2. Adjacent, characterization PCR tube(southward) with the ethanol-resistant marker.

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

   • X buffer (usually supplied by the manufacturer of the DNA polymerase; may contain 15 mM MgCl₂). Add v μl of 10X buffer per reaction.

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

   • 1.5 mM Mg^two+. Add only if it is not present in the 10X buffer or as needed for PCR optimization. For example, to obtain the 4.0 mM Mg²⁺ required for optimal amplicon production of a conserved 566 bp Dna segment constitute in an uncharacterized Mycobacteriophage add viii μl of 25 mM MgCl_two to the reaction (Figure 3).

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

   • Add x^four to 10⁷ molecules (or nigh one to 1000 ng) Dna template. Add 0.5 μl of 2ng/μl genomic Mycobacteriophage DNA.

   • Add 0.5 to 2.5 units of DNA polymerase per l μl reaction (Come across manufacturers recommendations) For example, add 0.5 μl of Sigma 0.v Units/μl Taq Deoxyribonucleic acid polymerase.

   • Add together Q.S. sterile distilled water to obtain a 50 μl final volume per reaction as pre-determined in the tabular array of reagents (Q.South. is a Latin abridgement for quantum satis significant the amount that is needed). Thus, 33 μl per reaction is required to bring the volume up to fifty μl. Nevertheless, it should be noted that water is added first just requires initially making a table of reagents and determining the volumes of all other reagents added to the reaction.

iv. Basic PCR Protocol

1. Place a 96 well plate into the ice bucket equally a holder for the 0.2 ml thin walled PCR tubes. Allowing PCR reagents to be added into cold 0.two ml sparse walled PCR tubes will help foreclose nuclease activeness and nonspecific priming.

2. Pipette the following PCR reagents in the post-obit order into a 0.2 ml thin walled PCR tube (Figure 4): Sterile Water, 10X PCR buffer, dNTPs, MgCl₂, primers, and template DNA (Run into Table 1). Since experiments should have at least a negative control, and perhaps a positive control, information technology is beneficial to set a Main Mix in a ane.eight ml microcentrifuge tube (See caption in Notes).

3. In a separate 0.2 ml thin walled PCR tubes (Effigy 4) add all the reagents with the exception of template DNA for a negative control (increase the water to recoup for the missing volume). In addition, another reaction (if reagents are bachelor) should contain a positive control using template Deoxyribonucleic acid and or primers previously known to amplify under 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 past pipetting upwards and down at least xx times. The micropipettor should be set to nearly half the reaction book of the master mix when mixing, and intendance should be taken to avoid introducing bubbles.

5. Put caps on the 0.2 ml sparse walled PCR tubes and place them into the thermal cycler (Figure 5). In one case the hat to the thermal cycler is firmly closed start the plan (see Table 2).

6. When the program has finished, the 0.ii ml thin walled PCR tubes may be removed and stored at four °C. PCR products can exist detected by loading aliquots of each reaction into wells of an agarose gel and so 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 exist visualized with a UV illuminator.

Notes:

1. When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions (i.e., Master Mix). Usually the cocktail contains a solution of Deoxyribonucleic acid polymerase, dNTPs, reaction buffer, and water assembled into a one.viii ml microcentrifuge tube. The amount of each reagent added to the Master Mix is equivalent to the total number of reactions plus 10% rounded up to the nearest whole reaction. For case, if there are 10 ten 0.1 = 1 reaction, so (x + 1) x 5 μl 10X buffer equals 55 μl of 10X buffer for the Master Mix. The reagents in the Master Mix are mixed thoroughly by gently pumping the plunger of a micropipettor up and down about 20 times as described above. Each PCR tube receives an aliquot of the Principal Mix to which the DNA template, any required primers, and experiment-specific reagents are so added (run into Tables 1 and seven).

2. The following website offers a computer for determining the number of copies of a template DNA (http://www.uri.edu/inquiry/gsc/resources/cndna.html). The total number of copies of double stranded DNA may exist calculated using the following equation: Number of copies of DNA = (Deoxyribonucleic acid corporeality (ng) ten 6.022x10²³) / (length of Dna x 1x10⁹ ng/ml x 650 Daltons) Calculating the number of copies of Deoxyribonucleic acid is used to determine how much template is needed per reaction.

3. False positives may occur equally a consequence of carry-over from another PCR reaction which would exist visualized as multiple undesired products on an agarose gel after electrophoresis. Therefore, it is prudent to use proper technique, include a negative control (and positive control when possible).

4. While ethidium bromide is the most common stain for nucleic acids there are several safer and less toxic alternatives. The post-obit website describes several of the alternatives including Methylene Blue, Crystal Violet, SYBR Rubber, and Gel Ruby along with descriptions of how to utilise and notice the concluding production (http://bitesizebio.com/articles/ethidium-bromide-the-alternatives/).

5. While near modern PCR machines use 0.2 ml tubes, some models may require reactions in 0.5 ml tubes. See your thermal cyclers manual to make up one's mind the appropriate size tube.

6. Setting Up Thermal Cycling Weather condition

1. PCR thermal cyclers rapidly heat and cool the reaction mixture, assuasive 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 production. Cycling times are calculated based on the size of the template and the GC content of the Deoxyribonucleic acid. The general formula starts with an initial denaturation footstep at 94 °C to 98 °C depending on the optimal temperature for DNA polymerase activeness and Chiliad-C content of the template Deoxyribonucleic acid. A typical reaction volition commencement with a one infinitesimal denaturation at 94 °C. Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. Ane method, known every bit hot-start PCR, drastically extends the initial denaturation fourth dimension from three minutes upward to 9 minutes. With hot-kickoff PCR, the Deoxyribonucleic acid polymerase is added after the initial exaggerated denaturation step is finished. This protocol modification avoids likely inactivation of the Dna polymerase enzyme. Refer to the Troubleshooting section of this protocol for more information about hot kickoff PCR and other alternative methods.

2. The next pace is to set the thermal cycler to initiate the first of 25 to 35 rounds of a iii-stride temperature bike (Table 2). While increasing the number of cycles above 35 will result in a greater quantity of PCR products, too many rounds frequently results in the enrichment of undesirable secondary products. The 3 temperature steps in a single cycle accomplish three tasks: the first step denatures the template (and in later cycles, the amplicons as well), the 2d step allows optimal annealing of primers, and the third step permits the DNA polymerase to demark to the DNA template and synthesize the PCR product. The duration and temperature of each step inside a cycle may exist altered to optimize production of the desired amplicon. The time for the denaturation step is kept equally brusk as possible. Usually 10 to 60 seconds is sufficient for nearly DNA templates. The denaturation time and temperature may vary depending on the Yard-C content of the template DNA, as well as the ramp rate, which is the fourth dimension it takes the thermal cycler to modify from one temperature to the next. The temperature for this footstep is usually the same equally that used for the initial denaturation phase (step #1 above; e.k., 94 °C). A 30 second annealing stride follows within the wheel at a temperature set about v °C beneath the credible T_thou of the primers (ideally between 52 °C to 58 °C). The bicycle concludes with an elongation footstep. The temperature depends on the Deoxyribonucleic acid polymerase selected for the experiment. For example, Taq DNA polymerase has an optimal elongation temperature of seventy °C to fourscore °C and requires ane minute to elongate the first 2 kb, so requires an extra minute for each additional one 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 use in PCR and primer extension reactions that require high fidelity and requires 2 minutes for every ane kb to exist amplified. See manufacturer recommendations for exact elongation temperatures and elongation time indicated for each specific DNA polymerase.

3. The final phase of thermal cycling incorporates an extended elongation menstruum of five minutes or longer. This last step allows synthesis of many uncompleted amplicons to finish and, in the case of Taq DNA polymerase, permits the improver of an adenine residuum to the 3' ends of all PCR products. This modification is mediated by the final transferase activity of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that crave a 3'-overhang.

4. Termination of the reaction is achieved by chilling the mixture to 4 °C and/or by the addition of EDTA to a terminal concentration of 10 mM.

7. Of import Considerations When Troubleshooting PCR

If standard PCR conditions do not yield the desired amplicon, PCR optimization is necessary to reach better results. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables (e.yard., reagent concentrations, cycling conditions) that affect the outcome 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 too stringent, no product volition exist produced. Troubleshooting PCR reactions may be a frustrating effort at times. However, conscientious analysis and a good understanding of the reagents used in a PCR experiment can reduce the amount of time and trials needed to obtain the desired results. Of all the considerations that impact PCR stringency, titration of Mg²⁺ and/or manipulating annealing temperatures probable will solve nigh problems. However, before irresolute anything, be sure that an erroneous result was not due to homo mistake. Start past confirming all reagents were added to a given reaction and that the reagents were not contaminated. Also accept note of the erroneous issue, and enquire the following questions: Are primer dimers visible on the gel after electrophoresis (these run equally small-scale bands <100 b near the lesser of the lane)? Are there not-specific products (bands that migrate at a different size than the desired product)? Was there a lack of whatever product? Is the target DNA on a plasmid or in a genomic Dna extract? As well, it is wise to analyze the Yard-C content of the desired amplicon.

1. First determine if whatever of the PCR reagents are catastrophic to your reaction. This can be accomplished past preparing new reagents (e.1000., fresh working stocks, new dilutions), and then systematically adding one new reagent at a fourth dimension to reaction mixtures. This process will decide which reagent was the culprit for the failed PCR experiment. In the example of very former DNA, which often accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may aid alleviate the problem.

2. Primer dimers can form 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 appear on the agarose gel. Start by altering the ratio of template to primer; if the primer concentration is in extreme backlog over the template concentration, so the primers will be more likely to anneal to themselves or each other over the Deoxyribonucleic acid template. Adding DMSO and or using a hot start thermal cycling method may resolve the problem. In the terminate it may be necessary to pattern 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 appropriate to choose PCR conditions that increase stringency. A smear of various sizes may as well upshot from primers designed to highly repetitive sequences when amplifying genomic Dna. However, the same primers may amplify a target sequence on a plasmid without encountering the same problem.

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

5. If the G-C content has non been analyzed, it is time to do so. PCR of 1000-C rich regions (GC content >60%) pose some of the greatest challenges to PCR. Nonetheless, in that location are many additives that have been used to help convalesce the challenges.

eight. Manipulating PCR Reagents

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

1. Magnesium salt Mg²⁺ (concluding reaction concentration of 0.5 to 5.0 mM) Thermostable Dna polymerases crave the presence of magnesium to deed as a cofactor during the reaction procedure. Irresolute the magnesium concentration is 1 of the easiest reagents to dispense with possibly the greatest impact on the stringency of PCR. In general, the PCR product yield will increase with the addition of greater concentrations of Mg²⁺. Still, increased concentrations of Mg^two+ volition also decrease the specificity and allegiance of the Dna polymerase. Nearly manufacturers include a solution of Magnesium chloride (MgCl₂) forth with the DNA polymerase and a 10X PCR buffer solution. The x X PCR buffer solutions may comprise fifteen mM MgCl₂, which is enough for a typical PCR reaction, or it may exist added separately at a concentration optimized for a detail reaction. Mg^two+ is non really consumed in the reaction, simply the reaction cannot proceed without it being present. When there is too much Mg²⁺, it may prevent consummate denaturation of the DNA template past stabilizing the duplex strand. Too much Mg²⁺ also tin can stabilize spurious annealing of primers to wrong template sites and decrease specificity resulting in undesired PCR products. When in that location is not enough Mg²⁺, the reaction will not keep, resulting in no PCR product.

2. Potassium salt K⁺ (final reaction concentration of 35 to 100 mM) Longer PCR products (10 to forty kb) benefit from reducing potassium salt (KCl) from its normal l mM reaction concentration, often in conjunction with the addition of DMSO and/or glycerol. If the desired amplicon is below 1000 bp and long non-specific products are forming, specificity may be improved past titrating KCl, increasing the concentration in 10 mM increments upwards 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 5'-triphosphates (dNTPs) tin cause bug for PCR if they are not at the advisable equivalent concentrations (i.e., [A] = [T] = [C] = [G]) and/ or due to their instability from repeated freezing and thawing. The usual dNTP concentration is 50 μM of EACH of the four dNTPs. Withal, 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 can actually inhibit PCR. However, for longer PCR-fragments, a college dNTP concentration may be required. A large modify in the dNTP concentration may necessitate a corresponding change in the concentration of Mgⁱⁱ⁺.

4. Thermal stable Deoxyribonucleic acid polymerases PCR enzymes and buffers associated with those enzymes have come up a long style since the initial Taq Dna polymerase was first employed. Thus, choosing an appropriate enzyme tin can be helpful for obtaining desired amplicon products. For case the employ of Taq Dna polymerase may exist preferred over Pfu Deoxyribonucleic acid polymerase if processivity and/or the add-on of an adenine remainder to the three' ends of the PCR product is desired. The addition of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. Even so, if fidelity is more important an enzyme such as Pfu may be a better pick. Several articles accept an array of specific DNA polymerases designed for specialized needs. Accept a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the advisable Deoxyribonucleic acid polymerase. Most articles have tables that aid DNA polymerase selection by listing characteristics such as allegiance, yield, speed, optimal target lengths, and whether information technology is useful for G-C rich distension or hot start PCR.

5. Template DNA Dna 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 50 μg/ml of double stranded DNA or 40 μg/ml for either RNA or single stranded Deoxyribonucleic acid at an OD₂₆₀ =1.0. DNA extraction contaminants are common inhibitors in PCR and should be advisedly avoided. Common DNA 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 guess of the purity of extracted Dna. Ideally, the ratio of OD_260/280 is between i.viii and 2.0. Lower OD_260/280 is indicative of protein and/ or solvent contamination which, in all probability, will be problematic for PCR. In addition to the quality of template Dna, optimization of the quantity of DNA may greatly benefit the outcome of a PCR experiment. Although it is user-friendly to decide the quantity in ng/μl, which is frequently the output for modern nanospectrophotometers, the relevant unit of measurement for a successful PCR experiment is the number of molecules. That is, how many copies of DNA template comprise a sequence complementary to the PCR primers? Optimal target molecules are between x^four to 10⁷ molecules and may be calculated as was described in the notes in a higher place.

9. Additive Reagents

Condiment reagents may yield results when all else fails. Understanding the reagents and what they are used for is disquisitional in determining which reagents may exist virtually constructive in the acquisition of the desired PCR product. Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may bear on the usable concentration of another reagent. In add-on to the reagents listed beneath, proprietary commercially available additives are available from many biotechnology companies.

10. Additives That Benefit G-C Rich Templates

1. Dimethylsulfoxide (final reaction concentration of 1-10% DMSO) In PCR experiments in which the template DNA is particularly One thousand-C rich (GC content >60%), calculation DMSO may enhance the reaction by disrupting base pairing and effectively lowering the T_grand. Some T_grand calculators include a variable entry for calculation the concentration of DMSO desired in the PCR experiment. However, calculation more 2% DMSO may crave adding more Deoxyribonucleic acid polymerase every bit information technology has been demonstrated to inhibit Taq DNA polymerase.

2. Formamide (concluding reaction concentration of ane.25-10%) Similar DMSO, formamide also disrupts base pairing while increasing the stringency of primer annealing, which results in less non-specific priming and increased distension efficiency. Formamide also has been shown to be an enhancer for G-C rich templates.

3. 7-deaza-2'-deoxyguanosine 5'-triphosphate (last reaction concentration of dc^sevenGTP; three dc^sevenGTP:i dGTP fifty μM) Using three parts, or 37.5 μM, of the guanosine base analog dc⁷GTP in conjunction with 1 part, or 12.5 μM, dGTP will destabilize formation of secondary structures in the product. As the amplicon or template DNA is denatured, information technology will ofttimes form secondary structures such as hairpin loops. Incorporation of dc⁷GTP into the Deoxyribonucleic acid amplicon will prohibit germination of these aberrant structures.

Note:

dc⁷GTP attenuates the betoken of ethidium bromide staining which is why information technology is used in a 3:one ratio with dGTP.

1. Betaine (final reaction concentration of 0.5M to ii.5M) Betaine (Northward,N,North-trimethylglycine) is a zwitterionic amino acid analog that reduces and may even eliminate the Deoxyribonucleic acid melting temperature dependence on nucleotide composition. It is used as an additive to aid PCR distension of Thousand-C rich targets. Betaine is ofttimes employed in combination with DMSO and can greatly raise the chances of amplifying target DNA with loftier Grand-C content.

11. Additives That Help PCR in the Presence of Inhibitors

1. Non ionic detergents part to suppress secondary structure formation and help stabilize the Dna polymerase. Non ionic detergents such equally Triton Ten-100, Tween xx, or NP-40 may exist used at reaction concentrations of 0.one to ane% in order to increase amplicon production. However, concentrations above 1% may be inhibitory to PCR. The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. However, their employ will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols.

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

12. Modifications to Cycling Weather

1. Optimizing the annealing temperature will enhance whatever PCR reaction and should exist considered in combination with other additives and/ or along with other modifications to cycling conditions. Thus, in order to calculate the optimal annealing temperature the following equation is employed: T_a ^OPT = 0.3 T_g ^Primer + 0.7 T_grand ^Production -14.9 T_m ^Primer is calculated every bit the T_m of the less stable pair using the equation: T_m ^Primer = ((ΔH/(ΔS+R x ln(c/4)))-273.15 + 16.half dozen log[K⁺] Where ΔH is the sum of the nearest neighbour enthalpy changes for hybrids; ΔS is the sum of the nearest neighbor entropy changes; R is the Gas Constant (1.99 cal K-ane mol-one); C is the primer concentration; and [K⁺] is the potassium concentration. The latter equation can be computed using 1 of the T_m calculators listed at the following website: http://poly peptide.bio.puc.cl/cardex/servers/melting/sup_mat/servers_list.html T_m ^Product is calculated as follows: T ₁₀₀₀ ^Production = 0.41(%G-C) + xvi.6 log [K⁺] - 675/production length For nigh PCR reactions the concentration of potassium ([M⁺]) is going to be 50 mM.

2. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically (Tabular array 4). This modification can be incorporated with or without other modifications to cycling conditions. Moreover, it is oft used in conjunction with additives for temperamental amplicon formation. In fact, hot commencement PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and allegiance of the reaction. The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result every bit a consequence of setting upwardly the reaction below the T_m. Thus, a typical hot start reaction heats the sample to a temperature above the optimal T_grand, at to the lowest degree to 60 °C before whatsoever amplification is able to occur. In general, the Dna 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. There are several methods which let the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax bulwark, anti-Deoxyribonucleic acid polymerase antibodies, and accessory proteins. Alternatively, the DNA polymerase may simply exist added to the reaction later the initial denaturation cycle is consummate.

3. Touchdown PCR (TD-PCR) is intended to accept some of the gauge work out of the T_k calculation limitations by bracketing the calculated annealing temperatures. The concept is to design 2 phases of cycling weather (Table 5). The first phase employs successively lower annealing temperatures every second cycle (traditionally i.0 °C), starting at 10 °C above and finishing at the calculated T_chiliad or slightly beneath. Stage 2 utilizes the standard 3-step conditions with the annealing temperature set at 5 °C below the calculated T_thousand for another 20 to 25 cycles. The function of the first stage should alleviate mispriming, conferring a 4-fold advantage to the correct product. Thus, later on 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 start phase of priming. As an case, the kickoff phase lowers annealing temperatures every second cycle past 3 °C, starting at x °C above and finishing at 2 °C below the calculated T_m. Like TD-PCR, stage 2 utilizes the standard 3-step atmospheric condition with the annealing temperature set at v °C below the calculated T_m for another 20 to 25 cycles. This would allow the correct product a 256-fold advantage over false priming products.

   • Slowdown PCR is but a modification of TD-PCR and has been successful for amplifying extremely One thousand-C rich (above 83%) sequences (Table 6). The concept takes into account a relatively new feature associated with mod thermal cyclers, which allows aligning of the ramp speed likewise as the cooling rate. The protocol likewise utilizes dc⁷GTP to reduce ii °structure formation that could inhibit the reaction. The ramp speed is lowered to 2.5 °C southward^-1 with a cooling charge per unit of 1.v °C s^-one for the annealing cycles. The first phase starts with an annealing temperature of 70 °C and reduces the annealing temperature past ane °C every three rounds until it reaches 58 °C. The second stage and then continues with an annealing temperature of 58 °C for an additional fifteen cycles.

4. Nested PCR is a powerful tool used to eliminate spurious products. The utilize of nested primers is particularly helpful when at that place are several paralogous genes in a single genome or when in that location is low re-create number of a target sequence within a heterogeneous population of orthologous sequences. The basic procedure involves two sets of primers that dilate a single region of DNA. The outer primers straddle the segment of interest and are used to generate PCR products that are often non-specific in 20 to 30 cycles. A small aliquot, usually about v μl from the first fifty μl reaction, is then used as the template DNA for some other 20 to thirty rounds of amplification using the 2d set of primers that anneal to an internal location relative to the beginning set.

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.

thirteen. Representative Results

Representative PCR results were generated past following the bones 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 3-footstep PCR protocol outlined in Table 2 was employed for all three experiments described below.

Earlier setting upward the PCR experiment, the genomic DNA from both S. cerevisiae and the Mycobacteriophage were quantified and diluted to a concentration that would let between x⁴ and 10^vii molecules of DNA per reaction. The working stocks were prepared as follows. A genomic yeast Deoxyribonucleic acid grooming yielded 10^four ng/μl. A dilution to 10 ng/μl was generated past adding 48 μl into 452 μl of TE pH viii.0 buffer. Since the S. cerevisiae genome is near 12.five Mb, 10 ng contain 7.41 Ten 10^v molecules. The genomic Mycobacteriophage DNA preparation yielded 313 ng/μl. A dilution to 2 ng/μl was generated by adding 6.4 μl into 993.6 μl of TE pH 8.0 buffer. This phage Deoxyribonucleic acid is about 67 Kb. Thus, one ng contains ii.73 Ten 10⁷ molecules, which is at the upper limit of Deoxyribonucleic acid generally used for a PCR. The working stocks were and so used to generate the Master Mix solutions outlined in Table 7. Experiments varied cycling conditions as described below.

In Effigy 3a, genomic DNA from Due south. cerevisiae was used every bit a template to amplify the GAL3 gene, which encodes a poly peptide involved in galactose metabolism. The goal for this experiment was to determine the optimal Mgⁱⁱ⁺ 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 five.0 mM. As shown in the figure, a PCR product of the expected size (2098 bp) appears starting at a Mg^two+ concentration of two.5 mM (lane half dozen) with an optimal concentration at 4.0 mM (lane 9). The recommended concentration provided by the manufacturer was i.5 mM, which is the amount provided in typical PCR buffers. Perhaps surprisingly, the necessary concentration needed for product formation in this experiment exceeded this amount.

A unlike DNA template was used for the experiment presented in Figure 3b. Genomic DNA from a Mycobacteriophage was used to amplify a conserved 566 bp DNA segment. Like the previous experiment, the optimal Mg²⁺ concentration had to be determined. Equally shown in Figure 3b, amplification of the desired PCR production requires at least 2.0 mM Mg^two+ (lane 5). While there was more variability in the amount of product formed at increasing concentrations of MgCl_two, the most PCR product was observed at 4 mM Mgⁱⁱ⁺ (lane 9), the same concentration observed for the yeast GAL3 factor.

Notice that in the experiments presented in Figures 3A and 3B, a discrete band was obtained using the cycling weather condition idea 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 i minute at 72 °C for thirty cycles. The last five infinitesimal extension was then done at 72 °C. For the 3rd experiment presented in Figure 3c, three changes were fabricated to the cycling conditions used to amplify the yeast GAL3 factor. First, the annealing temperature was reduced to a sub-optimal temperature of 58 °C. 2d, the extension fourth dimension was extended to 1 minute and thirty seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal distension conditions (i.east., reducing the stringency of the reaction) on a PCR experiment. As shown in Figure 3c, what was a detached 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 Mg²⁺ is required to class an amplicon.

All three experiments illustrate that when Mg²⁺ concentrations are too low, there is no amplicon production. 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 discreet amplicon corresponding to the expected size. The results show the importance of performing PCR experiments at a sufficiently loftier stringency (e.k., discreet bands versus a smear). Moreover, the experiments indicate that changing ane parameter can influence another parameter, thus affecting the reaction outcome.

Reagent	Concentration of stock solutions	Book	13X ** Master Mix	Final Concentration
Sterile H2O		Q.Due south. to fifty μl	Q.S. to 650 μl
PCR Buffer	10X	5 μl	65 μl	1X
dNTP's	x mM	ane μl	thirteen μl	200 μM
MgCl2	25 mM	3 μl	39 μl	1.5 mM
Forward Primer	20 μM = 20 pmol/μl	1 μl	13 μl	20 pmol
Reverse Primer	20 μM = 20 pmol/μl	ane μl	xiii μl	20 pmol
Template DNA	Variable	Variable	Variable	~105 Molecules
Taq DNA Polymerase	5 Units/μl*	0.five μl	6.5 μl	2.5 Units
				50 μl/Reaction

Table 1. PCR reagents in the order they should be added.

*Units may vary betwixt manufacturers

** Add all reagents to the Chief Mix excluding any in need of titration or that may be variable to the reaction. The Master Mix depicted in the in a higher place tabular array is calculated for 11 reactions plus 2 actress reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube.

	Standard 3-pace PCR Cycling
Cycle footstep	Temperature	Time	Number of Cycles
Initial Denaturation	94 °C to 98 °C	1 minute	1
Denaturation Annealing Extension	94 °C 5 °C beneath Tm 70 °C to 80 °C	10 to sixty seconds thirty seconds Amplicon and Dna polymerase dependent	25-35
Final Extension	70 °C to 80 °C	5 minutes	one
Hold*	four °C	∞	one

Table 2. Standard three-step PCR Cycling.

* Most thermal cyclers take the ability to pause at 4°C indefinitely at the end of the cycles.

	2-step PCR Cycling
Bicycle step	Temperature	Time	Number of Cycles
Initial Denaturation	94 °C to 98 °C	one infinitesimal	1
Denaturation Annealing/Extension	94 °C 70 °C to 80 °C	x to 60 seconds Amplicon and Dna polymerase dependent	25-35
Final Extension	seventy °C to 80 °C	5 minutes	1

Table 3. 2-step PCR Cycling.

	Hot Start PCR Cycling
Cycle step	Temperature	Time	Cycles
Initial Denaturation	sixty °C to 95 °C	5 minute then add together Deoxyribonucleic acid polymerase	1
Denaturation Annealing Extension	94 °C 5 °C beneath Tm 70 °C to 80 °C	10 to 60 seconds xxx seconds Amplicon and DNA polymerase dependent	25-35
Final Extension	70 °C to 80 °C	5 minutes	1

Table 4. Hot Commencement PCR Cycling.

	Touchdown PCR Cycling
Bike step	Temperature	Fourth dimension	Cycles
Initial Denaturation	94 °C to 98 °C	1 minute	one
Denaturation Annealing Extension	94 °C Ten =10 °C above Tg 70 °C to lxxx °C	10 to 60 seconds 30 seconds Amplicon and DNA polymerase dependent	2
Denaturation Annealing Extension	94 °C X-1 °C reduce one °C every other bicycle 70 °C to 80 °C	10 to 60 seconds 30 seconds Amplicon and polymerase dependent	28
Denaturation Annealing Extension	94 °C 5 °C below 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 5. Touchdown PCR Cycling.

	Slowdown PCR Cycling
Cycle step	Temperature	Time	Cycles
Initial Denaturation	94 °C to 98 °C	1 infinitesimal	1
Denaturation Annealing Extension	94 °C X °C =10 °C in a higher place Tthousand 70 °C to 80 °C	10 to 60 seconds 30 seconds Amplicon and polymerase dependent	two
Denaturation Annealing Extension	94 °C Ten-1 °C reduce 1 °C every other cycle lxx °C to 80 °C*	10 to 60 seconds 30 seconds Amplicon and polymerase dependent	28
Denaturation Annealing Extension	94 °C 5 °C below Tm seventy °C to 80 °C	10 to 60 seconds 30 seconds Amplicon and polymerase dependent	20-25
Final Extension	lxx °C to 80 °C	5 minutes	1

Table half dozen. Slowdown PCR Cycling.

*For slowdown PCR, the ramp speed is lowered to ii.5 °C due south^-ane with a cooling rate of 1.v °C s^-1 for the annealing cycles.

	Stock Solution	Book added to l μl reaction	thirteen X Yeast Master Mix	13 X Phage Master Mix	Last Concentration
Sterile HiiO	q.southward. to 50 μl = 31 μl or 30.five	q.s. to 650 μl = 396.5	q.s. to 520 μl = 403 μl
PCR Buffer	10X	5 μl	65 μl	65 μl	1X
dNTP's	10 mM	1 μl	13 μl	xiii μl	200 μM
MgClii	Titration	Added to each reaction	Added to each reaction	Added to each reaction	Variable see titration
Forward Primer	20 μM = xx pmol/μl	1 μl	13 μl	xiii μl	20 pmol
Reverse Primer	20 μM = 20 pmol/μl	1 μl	thirteen μl	13 μl	20 pmol
Template DNA	ii ng/μl phage or 10 ng/μl Yeast	0.v μl Phage or ane μl Yeast	6.5 μl	xiii μl	~xseven Molecules Phage or ~x5 Molecules Yeast
Polymerase	0.v Units/μl**	0.5 μl	6.5 μl	6.v μl	0.5 Units/Reaction
					xl μl + 10(Titration) μl/ Reaction
TITRATION
[MgClii]	0.00 mM	0.5 mM	1.0 mM	ane.5mM	ii.0 mM	two.5 mM	3.0 mM	three.v mM	4.0 mM	4.5 mM	5.0 mM
MgCl2	0.00 μl	1.00 μl	2.00 μl	three.0 μl	4.00 μl	five.00 μl	half-dozen.00 μl	7.00 μl	eight.00 μl	ix.00 μl	10.00 μl
H2O	10.00 μl	9.00 μl	8.00 μl	7.00 μl	6.00 μl	5.00 μl	four.00 μl	3.00 μl	2.00 μl	1.00 μl	0.00 μl

Table 7. Titration of Mg²⁺ used in Figure 3.

Figure 1. Mutual issues that arise with primers and iii-step PCR amplification of target DNA. (a) Self-annealing of primers resulting in formation of secondary hairpin loop structure. Annotation that primers exercise not e'er amalgamate at the extreme ends and may form smaller loop structures. (b) Primer annealing to each other, rather than the DNA template, creating primer dimers. In one case the primers anneal to each other they volition elongate to the primer ends. (c) PCR cycles generating a specific amplicon. Standard three-pace PCR cycling include denaturation of the template Deoxyribonucleic acid, annealing of primers, and extension of the target Deoxyribonucleic acid (amplicon) by DNA polymerase.

Effigy two. Water ice bucket with reagents, pipettes, and racks required for a PCR. (i.) P-200 pipette, (2.) P-yard pipette, (3.) P-xx pipette, (4.) P-10 pipette, (5.) 96 well plate and 0.2 ml thin walled PCR tubes, (6.) Reagents including Taq polymerase, 10X PCR buffer, MgCl_two, sterile water, dNTPs, primers, and template DNA, (7.) 1.eight ml tubes and rack.

Figure iii. Instance of a Mgⁱⁱ⁺ titrations used to optimize a PCR experiment using a standard 3-step PCR protocol. (a) S. cerevisiae Yeast genomic Dna was used every bit a template to amplify a 2098 bp GAL3 gene. In lanes 1 - vi, where the Mg²⁺ concentration is too low, at that place either is no product formed (lanes 1-5) or very little production formed (lane half-dozen). Lanes 7 - 11 represent optimal concentrations of Mg²⁺ for this PCR experiment equally indicated by the presence of the 2098 bp amplicon production. (b) An uncharacterized mycobacteriophage genomic DNA template was used to amplify a 566 bp amplicon. Lanes 1 - 4, the Mg^two+ concentration is likewise low, as indicated by the absence of product. Lanes 5 - xi represent optimal concentrations of Mg²⁺ for this PCR equally indicated past the presence of the 566 kb amplicon product. (c) . S. cerevisiae Yeast genomic Deoxyribonucleic acid was used every bit a template to amplify a 2098 bp GAL3 gene every bit 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 ane - 4, where the Mg²⁺ concentration is too depression, in that location is no product formed. Lanes v - 8 correspond optimal concentrations of Mg²⁺ for this PCR as seen by the presence of a smear and ring effectually the 2098 kb amplicon product size. Lanes 9 - 11 are indicative of excessively stringent weather with no product formed. (a-c) Lanes 12 is a negative control that did not comprise any template Dna. Lane Thou (marker) was loaded with NEB 1kb Ladder.

Effigy 4. Sterile tubes used for PCR. (i.) ane.8 ml tube (2.) 0.2 ml individual sparse walled PCR tube, (3.) 0.two ml strip sparse walled PCR tubes and caps.

Figure 5. Thermal cycler. Airtight thermal cycler left image. Correct image contains 0.2 ml sparse walled PCR tubes placed in the heating block of an open thermal cycler.

Discussion

PCR has become an indispensible tool in the biology arsenal. PCR has altered the course of science allowing biologists to yield power over genomes, and make hybrid genes with novel functions, allowing specific and authentic clinical testing, gaining insights into genomes and diversity, as well as simply cloning genes for further biochemical analysis. PCR application is limited only past the imagination of the scientist that wields its power. There are many books and papers that describe new specialized uses of PCR, and many more will be developed over the next generation of biology. Nonetheless, regardless of the anticipated approaches, the primal 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 idea and patience. The results shown in Figure 3 exemplify one of the major challenges when designing an optimization strategy for PCR. That is, as 1 parameter of PCR is changed, it may impact another. As an instance, if the initial PCR was carried out at the sub-optimal annealing temperature (58°C) with an optimal Mg²⁺ concentration of ii.0 mM, then the upshot would produce a smear as seen in Figure 3c. An effort to resolve the smear might involve setting upward 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 any product. Consequently, information technology is advisable to titrate reagents, rather than calculation ane concentration to a unmarried reaction, when troubleshooting spurious results. As well, 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 non minimize or abrogate aberrant results, titration of additives and /or changing the cycling condition protocols as described in Tables 2-vi may alleviate the problem. If all else fails, redesign the primers and try, attempt again.

Disclosures

I have null to disembalm.

Acknowledgments

Special cheers 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 too like to thanks Giancarlo Costaguta and Gregory Due south. Payne for supplying the yeast genomic DNA and primers to amplify the GAL3 gene. I would likewise like to give thanks Bhairav Shah for taking pictures of the lab equipment and reagents used to make figures 2 - four. Funding for this project was provided by HHMI (HHMI Grant No. 52006944).

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846334/

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