{"id":8653,"date":"2023-04-16T17:15:01","date_gmt":"2023-04-16T17:15:01","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=8653"},"modified":"2023-04-16T17:18:19","modified_gmt":"2023-04-16T17:18:19","slug":"pcr-troubleshooting-common-problems-and-solutions","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/pcr-troubleshooting-common-problems-and-solutions\/","title":{"rendered":"PCR Troubleshooting: Common Problems and Solutions"},"content":{"rendered":"<p>The process of <span id=\"urn:enhancement-85a5b73f-aaf1-4581-8a78-212d846ab366\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">Polymerase<\/span> Chain Reaction (PCR) is an innovative approach that allows for the amplification of samples to aid in their identification and analysis. In the fields of life sciences and medicine, <span id=\"urn:enhancement-ab10b745-fecd-4192-bce4-c09e72ffc226\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> has grown to become an influential tool. Its applications include the detection of pathogens, either specific or broad-spectrum, monitoring emerging infections, analyzing <span id=\"urn:enhancement-f76f9023-1ee1-4028-8797-f4f53319fd3d\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antimicrobial\">antimicrobial<\/span> resistance, screening for neonatal genetic disorders, and identifying genes linked to tumors.<\/p>\n<p><span id=\"urn:enhancement-1418adea-125a-41cd-a146-b934ab1ffb0f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> has had a remarkable impact on our comprehension of biology and medicine, as it provides quick and accurate results that are dependable. One of the primary advantages of <span id=\"urn:enhancement-beb2df06-e7d9-4b5f-91c9-623d45176d48\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is its speed, as it can quickly amplify target samples from a single source through the exponential growth of DNA fragments in the reaction. This can reduce the time taken to obtain results from days or weeks to just a few hours. Furthermore, <span id=\"urn:enhancement-ef8191a3-c4e4-494d-bcc6-1d5c5e0bfa1d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> assays are highly sensitive, making it possible to identify and analyze small samples such as single cells without requiring a vast amount of the specimen material.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-8654\" src=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/04\/5.1.jpg\" alt=\"\" width=\"485\" height=\"186\" \/><\/p>\n<p><span id=\"urn:enhancement-4bd06ecd-38e0-4d07-a610-5be05bdbeccf\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> has another benefit in that it provides excellent precision, particularly with techniques such as Real-Time Quantitative <span id=\"urn:enhancement-c5ff9a15-5251-49be-821b-6e5e0cdb4e06\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>, which can yield significant data on transcript levels or gene <span id=\"urn:enhancement-f23a32c1-5562-4594-9e99-469786f1ea6f\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/expression\">expression<\/span>. With various assay options at its disposal and the ability to design primers targeting specific genes, <span id=\"urn:enhancement-aeb36b63-710f-4962-98ee-b8611bedc139\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> can achieve highly specific detection of target cases, even in complex interactions that involve multiple genes from different species.<\/p>\n<p>Although <span id=\"urn:enhancement-3fc1dfb1-5508-4165-8989-a475a0d22a63\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is a powerful technique, sometimes <span id=\"urn:enhancement-3858c2d0-056d-411b-8a22-7cc4e374b834\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reactions can fail due to various reasons. Here are some common <span id=\"urn:enhancement-1b9f618e-fd91-4edd-a405-226c98cda92c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> troubleshooting problems and their solutions:<\/p>\n<ul>\n<li>\n<h4><strong>No Amplification or Low Yield:\u00a0<\/strong><\/h4>\n<\/li>\n<\/ul>\n<p>No amplification or low yield in <span id=\"urn:enhancement-18a1e909-7c10-4368-b7f4-7c5ed26b768c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is a common problem that researchers encounter during <span id=\"urn:enhancement-1af4b457-8c73-4e6b-87ee-c6a573dbac73\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> experiments. The first step in troubleshooting no amplification or low yield in <span id=\"urn:enhancement-78188acf-08f0-476b-87a1-783795c274ee\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is to confirm the presence of the DNA template. This can be done by measuring the concentration and purity of the DNA template using spectrophotometry or fluorometry. If the DNA concentration is low or the purity is poor, it can affect the success of the <span id=\"urn:enhancement-93df63cd-93dd-4a42-a8e5-6e07255c5d40\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction. In this case, the DNA template may need to be purified or concentrated before proceeding with the <span id=\"urn:enhancement-0503a119-0e26-43af-9a68-1b4818cf2351\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction.<\/p>\n<p>The next step is to optimize the <span id=\"urn:enhancement-3646d8e5-23a5-48e4-8097-2a875aac03ec\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> conditions. This involves adjusting the <span id=\"urn:enhancement-1e667bba-d166-4594-8a74-4164a6f7458f\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/tm\">annealing<\/span> temperature, the MgCl\u2082 concentration, and the reaction buffer. If the annealing temperature is too low or too high, it can result in non-specific amplification or no amplification, respectively. The MgCl\u2082 concentration affects the activity of the <span id=\"urn:enhancement-564311e4-9027-4d81-b444-ddd8ecca7ea6\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> enzyme and can affect the efficiency of the <span id=\"urn:enhancement-6f683208-9528-4072-b8c3-97c3feaeea83\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction. The reaction buffer contains salts and other components that are necessary for the <span id=\"urn:enhancement-73d12e5a-d896-42f9-8bb4-d1bfeee120e7\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction to proceed. If the buffer is suboptimal, it can affect the success of the <span id=\"urn:enhancement-3e3db869-0bbf-4bed-9d47-377e571e3dcf\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction.<\/p>\n<p>Also, the amount of enzyme and dNTPs can affect the success of the <span id=\"urn:enhancement-f6ef6b14-04f0-425e-8ba9-bafea8de15ff\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction. If the amount of enzyme or dNTPs is too low, it can result in no amplification or low yield. Increasing the amount of enzyme or dNTPs can improve the efficiency of the <span id=\"urn:enhancement-7113b845-e3c6-451f-ba73-8a302827ceae\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction.<\/p>\n<ul>\n<li>\n<h4><strong>Non-Specific Products<\/strong><\/h4>\n<\/li>\n<\/ul>\n<p>Non-specific amplification occurs when the primers bind to unintended regions of the template DNA, resulting in the amplification of nonspecific products.<\/p>\n<p>Some polymerases exhibit low activity when exposed to room temperature or 4 \u00b0C. During the mixing of different reaction components, primers may anneal non-specifically, leading to the enzyme elongating these primers and generating a series of non-specific products. To prevent these non-specific products, hot-start polymerases can be employed. Hot-start enzymes can be created through various techniques, such as manual or physical separation, antibodies, or chemical modification. In the manual technique, one of the reaction mixture components, such as <span id=\"urn:enhancement-85c46d80-dd41-4ebe-b16f-7f197769f869\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/mg2\">Mg2<\/span>+, is added to the tube after the temperature exceeds 70 \u00b0C. Physical separation involves the use of a barrier, such as a wax plug, to separate reaction components until the temperature rises above 75 \u00b0C, at which point the wax melts and the components are mixed. Another method involves inactivating polymerases through heat-sensitive antibodies or heat-labile blocking groups that are added to specific <span id=\"urn:enhancement-3ca76eb4-48a2-4de1-860b-b876ef23c904\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/amino-acid\">amino acids<\/span>. At higher temperatures, the blocking groups are removed, and the enzyme is activated.<\/p>\n<ul>\n<li>\n<h4><strong>Primer-Dimer Formation:\u00a0<\/strong><\/h4>\n<\/li>\n<\/ul>\n<p>Primer-dimer formation can occur when the primers anneal to each other due to the complementarity between their sequences, resulting in a self-priming event. These self-priming events can be promoted by high primer concentrations, high annealing temperatures, and long annealing times. Primer-dimer formation is a problem in <span id=\"urn:enhancement-77f6f392-cf77-4584-a776-00cd2765b711\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> because it can reduce the yield of the target <span id=\"urn:enhancement-fac4bff5-eee5-45e0-bfb0-c8c239d1ede5\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/dna-sequence\">DNA sequence<\/span>, leading to false negative results, and also can create additional, unwanted products that can interfere with downstream analysis.<\/p>\n<p>There are several ways to prevent the primer-dimer formation in <span id=\"urn:enhancement-867b5749-ae0f-4cb2-b0de-d5fd69772eb4\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span>. The first is to optimize the <span id=\"urn:enhancement-dee4d3a9-9d44-4e69-9a1a-fe62c1811515\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> conditions, such as annealing temperature, primer concentration, and annealing time. Increasing the annealing temperature can reduce the likelihood of primer-dimer formation, but it can also decrease the efficiency of primer annealing to the target DNA.<\/p>\n<p>Another approach is to design the primers carefully. Primers should be designed with <span id=\"urn:enhancement-dff0d43f-cfb6-4cd6-8e39-66ce00e4a9a7\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/specificity\">specificity<\/span> and a minimum amount of complementarity between themselves. Primers should also be checked for secondary structures, which can promote self-priming events. Software programs are available to help design primers with <span id=\"urn:enhancement-a68d79cf-aeb9-446f-b346-e7aa2c58395e\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/specificity\">specificity<\/span> and minimize the potential for self-priming.<\/p>\n<ul>\n<li>\n<h4><strong>Inhibition of PCR:\u00a0<\/strong><\/h4>\n<\/li>\n<\/ul>\n<p>The inhibitors of the <span id=\"urn:enhancement-1cb11f29-48f7-49dd-8b53-761a05b7c0bb\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reaction refer to diverse organic and inorganic compounds that may obstruct DNA <span id=\"urn:enhancement-3fa03d40-b951-46ed-b4a5-9ff468fdedc0\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> directly (by causing <span id=\"urn:enhancement-33763358-869b-441f-b3d0-22811d4741d4\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span> degradation or obstructing the polymerase&#8217;s active center) or indirectly (by blocking the active center for cofactors such as magnesium ions) and\/or interact with the <span id=\"urn:enhancement-8e09461c-9e81-4e56-8c74-d44ccf8bddb4\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/nucleic-acid\">nucleic acid<\/span> template.<\/p>\n<p>To minimize the effects of inhibition, several strategies can be employed. The first step is to optimize the <span id=\"urn:enhancement-71df21b1-b6a4-4d39-ae5d-d8c5ad74e11b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> conditions, including the concentrations of the reaction components and the cycling conditions. The concentrations of the reactants, such as the template DNA, primers, and DNA <span id=\"urn:enhancement-9abbc1d7-551f-4c19-a414-ad7c5fc931e7\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span>, should be optimized to maximize the efficiency of amplification while minimizing the effects of inhibition.<\/p>\n<p>Another approach is to use specialized <span id=\"urn:enhancement-9e125f95-17b4-4612-ba40-8af4baa633d1\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> additives, such as bovine <span id=\"urn:enhancement-82254272-8991-4750-a7fe-cc28b031c638\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/serum\">serum<\/span> albumin (BSA), to help overcome the effects of inhibition. BSA can help reduce the binding of inhibitors to the DNA <span id=\"urn:enhancement-511b2f23-6a68-42ce-95d1-4988a3b794bf\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/polymerase\">polymerase<\/span>, thereby improving its activity. Other additives, such as betaine, can also be used to reduce the effects of inhibition by destabilizing the secondary structure of the template DNA.<\/p>\n<ul>\n<li>\n<h4><strong>Uneven or Smeared bands:\u00a0<\/strong><\/h4>\n<\/li>\n<\/ul>\n<p>One of the primary causes of uneven or smeared bands in <span id=\"urn:enhancement-fad723d0-db8a-40cc-b6a2-016cadbbbcfb\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> is suboptimal <span id=\"urn:enhancement-a840a95d-d6b0-462e-bcd4-17c3f891da73\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> conditions. If the annealing temperature is too low, non-specific products can be generated, leading to smeared bands on the gel. If the extension time is too long, secondary products can be formed, which can contribute to smearing.The quality of the template DNA can also affect the appearance of the <span id=\"urn:enhancement-a396478b-42bf-4fb1-923d-c0e8459dfeb9\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> product on the gel. Degraded DNA can lead to the formation of shorter and larger fragments, which can contribute to smearing. Contaminants in the DNA sample can also interfere with <span id=\"urn:enhancement-2b7edfa6-46c1-49ec-8f18-bf2a2a60f9c4\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> amplification, leading to smearing.<\/p>\n<p>According to a study, the issue of <span id=\"urn:enhancement-a0046584-9312-4544-9994-32b9c4ec972c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> smears during genotyping is caused by the gradual accumulation of &#8220;amplifiable DNA contaminants&#8221; that are specific to the <span id=\"urn:enhancement-0006a6cc-05e1-4323-a417-2fb021dcef69\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> primers being used. As a result, previously reliable primers are no longer effective once smears appear. To address this problem, it is recommended to take preventive measures such as separating lab areas, reagents, and equipment for pre-<span id=\"urn:enhancement-9d7e75a7-ac93-4150-8058-95f764ffaf2f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> from post-<span id=\"urn:enhancement-5928608d-4d26-42ea-8c3e-86187808d78d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> to slow down contamination buildup. However, the most efficient solution is to switch to a new set of primers with different sequences that do not interact with the accumulated contaminants, thus completely resolving the smearing issue.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-8655\" src=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/04\/5.2.jpg\" alt=\"\" width=\"523\" height=\"234\" srcset=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/04\/5.2.jpg 523w, https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/04\/5.2-480x215.jpg 480w\" sizes=\"(min-width: 0px) and (max-width: 480px) 480px, (min-width: 481px) 523px, 100vw\" \/><\/p>\n<h4><strong>Important Considerations When Troubleshooting PCR<\/strong><\/h4>\n<ol>\n<li>If the standard conditions for <span id=\"urn:enhancement-42909e91-15c1-4adf-8514-6c89e284b03e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> do not produce the intended amplicon, it is necessary to optimize the <span id=\"urn:enhancement-f24b169a-281e-4be5-8988-eb1a4da7be94\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> process for better outcomes. The <span id=\"urn:enhancement-a9f362d8-32c2-42e5-b9b4-de5538c204a0\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/specificity\">specificity<\/span> of the reaction can be adjusted by modifying variables such as <span id=\"urn:enhancement-3efae767-d94f-4788-afb6-e487486f0ae4\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/reagent\">reagent<\/span> concentrations and cycling conditions to achieve the desired amplicon profile. For instance, if the reaction is not stringent enough, multiple spurious amplicons of varying lengths may be produced, whereas a reaction that is too stringent may not produce any product at all. Although troubleshooting <span id=\"urn:enhancement-d2fe90d3-0324-4258-b3ea-46837a0395aa\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reactions can be frustrating, careful analysis and a comprehensive understanding of the reagents used in the experiment can shorten the time and number of trials required to obtain the intended results.<\/li>\n<li>Among the factors that affect <span id=\"urn:enhancement-ffe2ade9-5e9e-488b-b3eb-f5fc9fcdce47\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> stringency, adjusting the concentration of <span id=\"urn:enhancement-c141c612-db08-4dd1-930e-72c3b7577439\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/mg2\">Mg2<\/span>+ and\/or modifying annealing temperatures are likely to address most issues. Nevertheless, it is important to ensure that any erroneous result was not caused by human error before making any changes. It is recommended to first verify that all necessary reagents were added to the reaction and that they were free from contamination.<\/li>\n<li>It is crucial to identify if any of the <span id=\"urn:enhancement-e7b96f34-9c91-4e6c-941d-c56ba8417e81\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> reagents are harmful to the reaction. To do so, prepare fresh working stocks or dilutions of the reagents and add them one at a time to the reaction mixture systematically. This procedure will help to pinpoint which specific <span id=\"urn:enhancement-957edfdd-77ed-46eb-b998-cff6b31fddc3\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/reagent\">reagent<\/span> caused the unsuccessful <span id=\"urn:enhancement-83f0a853-62da-497e-81ac-5c608dadcfcf\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> experiment.<\/li>\n<\/ol>\n<p>In general, <span id=\"urn:enhancement-e297ffa7-8025-48d1-aae9-b6a9b69416f1\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> has several inherent benefits over other techniques for observing minute changes at both macro and micro levels in cells and tissues that were previously difficult or costly to detect. However, any application involving <span id=\"urn:enhancement-f2fc75b1-b6f4-49ac-a773-d74648ba4d48\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/pcr\">PCR<\/span> must consider its pros and cons before attempting its use, as neglecting to do so could result in suboptimal outcomes due to its intricate nature.<\/p>\n<p><span style=\"text-decoration: underline;\"><strong>References<\/strong><\/span><\/p>\n<ol>\n<li><span>Polymerase Chain Reaction. Molecular Biology, 168\u2013198 | 10.1016\/B978-0-12-813288-3.00006-9<\/span><\/li>\n<li>\u015apibida, M., Krawczyk, B., Olszewski, M., &amp; Kur, J. (2016).\u00a0<em>Modified DNA polymerases for PCR troubleshooting. Journal of Applied Genetics, 58(1), 133\u2013142.<\/em>\u00a0doi:10.1007\/s13353-016-0371-4<\/li>\n<li><span>https:\/\/www.jove.com\/t\/3998\/polymerase-chain-reaction-basic-protocol-plus-troubleshooting<\/span><\/li>\n<li>Cause and solutions to the polymerase chain reaction smear problem in genotyping Dawn D. Han a , Rong Chen a , Erik R. Hill a , Michael R. Tilley a , Howard H. Gu a,b,\u00a4 a Department of Pharmacology, The Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, OH 43210, USA<\/li>\n<li>Wang M, Cai J, Chen J, Liu J, Geng X, Yu X, et al. PCR Techniques and Their Clinical Applications [Internet]. Polymerase Chain Reaction [Working Title]. IntechOpen; 2023. Available from: http:\/\/dx.doi.org\/10.5772\/intechopen.110220<\/li>\n<\/ol>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>The process of Polymerase Chain Reaction (PCR) is an innovative approach that allows for the amplification of samples to aid in their identification and analysis. In the fields of life sciences and medicine, PCR has grown to become an influential tool. Its applications include the detection of pathogens, either specific or broad-spectrum, monitoring emerging infections, [&hellip;]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-8653","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/8653","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=8653"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/8653\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=8653"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}