Proven tips for PCR primer design

Table Of Content

• Designing forward primer
• Necessity for the design of a reference sequence
• Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
• Improving the sensitivity of the RT-qPCR assay
• PCR Product Purification
• Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2

This virus has since been named severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) 1. SARS-CoV-2 is highly contagious and has rapidly expanded worldwide since its discovery. As of 12 May, 2020, a total of 4,088,848 cases of SARS-CoV-2 infection have been confirmed worldwide with 283,153 deaths 2, although this may be underestimated due to inadequate testing in many countries 3.

(PDF) A critical review of PCR primer design algorithms and crosshybridization case study - ResearchGate

(PDF) A critical review of PCR primer design algorithms and crosshybridization case study.

Posted: Wed, 24 Feb 2016 00:58:49 GMT [source]

Designing forward primer

We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers.

Necessity for the design of a reference sequence

A dilution to 2 ng/μl was generated by adding 6.4 μl into 993.6 μl of TE pH 8.0 buffer. 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. While this system is incredibly useful to amplify and precisely quantify a gene of interest, there are several obstacles that can lower primer efficiencies and jeopardize your experiment. While this system is incredibly useful to amplify and precisely quantify a gene of interest, there are several obstacles that can jeopardize your experiment. Overcoming low primer efficiency is the first step to a successful PCR, but how can you ensure your PCR product is concentrated and pure enough for downstream applications?

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In such a case, the target sequence can be inferred through the amino acid sequence. Degenerate primers can overcome the uncertainty of the target sequence due to the redundancy of the genetic code 39. The second is when degenerate primers are used to amplify targeted sequences across multiple genotypes by allowing variant primers to simultaneously amplify multiple sequences.

Steps

The single-target method is prone to lead to false negative results once a site in the target sequence is mutated. Thus, dual-target or multi-target detection can be additionally used to improve detection sensitivity. After multiple pairs of primers are designed, regardless of whether they are designed manually or by software, further verification is necessary. In silico analysis of primer and probe sequences is needed to analyse the specificity. Only by evaluating the diagnostic ability of the designed primers and probes on a number of samples, we can identify primers/probes with good sensitivity and specificity. Furthermore, the reaction conditions of the assay, such as the concentration of the primers and probes, Tm, and annealing temperature, must be optimized for maximum accuracy of the results.

Tips for PCR primer design Video - Technology Networks

Tips for PCR primer design Video.

Posted: Sat, 16 Nov 2019 08:00:00 GMT [source]

Improving the sensitivity of the RT-qPCR assay

Second, the extension time was extended to 1 minute and 30 seconds. The purpose was to demonstrate the effects of sub-optimal amplification conditions (i.e., reducing the stringency of the reaction) on a PCR experiment. As shown in Figure 3c, what was a discrete band 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 form an amplicon. Primers should generally run nucleotides long and can be optimized for target specificity. If your sample context is complex, specificity can be refined with your primer length.

PCR Product Purification

The maximum stability for the last five 3' bases of a left or right primer. The Tm calculation is controlled by Table of thermodynamic parameters and Salt correction formula (under advanced parameters). The default Table of thermodynamic parameters is "SantaLucia 1998" and the default Salt correction formula is "SantaLucia 1998" as recommended by primer3 program. Watch the protocol video below to learn how to design primers for PCR.

Niels Bohr liked to say it’s difficult to make predictions, especially about the future. Just so, when it comes to primer design there is no software simulation as complex as the biological reality of your assay. Basic PCR proceeds with a forward and reverse primer pair annealing to denatured, single-stranded DNA targets. This allows a DNA polymerase to synthesize complementary strands extending molecules in the 5´ to 3´ direction. In theory, 100% efficient PCR doubles the target amplicon at each cycle.

After designing the primer/probe following the general principle strategy, in silico analysis is important for specificity or exclusivity testing. For example, an alignment was performed with the oligonucleotide primer and probe sequences of the CDC 2019 nCoV RT-qPCR diagnostic panel with all publicly available nucleic acid sequences for 2019-nCoV in GenBank. It is best to confirm specificity again after primer probe design is completed, especially since the primers and probes of multiple RT-qPCR need to be mixed together. As a general principle, this is correct because the template can be paired to form the stem by itself, which may not be conducive to accurate PCR quantification. However, what we want to emphasize in this review is that, firstly, SARS-CoV-2 is an RNA virus, which is unstable and easily degraded by RNase A 64. In addition, during reverse transcription in the RT-qPCR assay, the temperature is mostly set to 55°C, and this temperature is conducive to the effective opening of RNA secondary structures 67.

Success simply may rely on changing the concentration of MgCl2, KCl, dNTPs, primers, template DNA, 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 one reagent should be manipulated at a time. Once all the parameters are set, clicking on ‘Get primers’ returns a list of potential forward and reverse primer sequences to choose from.

Moreover, the experiments indicate that changing one parameter can influence another parameter, thus affecting the reaction outcome. Sometimes there is no product at all, a low yield, the wrong amplicon, or unexpected PCR artifacts. PCR primers are interdependent with your sample type, target, reagents, and thermodynamic conditions. Establishing your PCR objective can guide your primer design and enzyme choices. It’s a good idea to review parameters and understand why primer design is such a critical, dependent variable in PCR.

Below are the main steps involved in qPCR primer design using the NCBI tool Primer-BLAST. This requires that the left or the right primers to span a junction that is just 3' of any such positions. For example, entering "50 100" would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position 100 and 101 (counting from 5' to 3'). You can also specify in the fields below the minimal number of nucleotides that the left or the right primer must have on either side of the junctions. This option is useful if you want a primer to a span specific junction on the template.

The expect value for a given BLAST match between a primer and a target is roughly proportional to the query sequence length given the same search database [6], but the query lengths used in the primer-only case and the template case are often very different. Therefore, there can be a large discrepancy in the expect values between the BLAST matches in the two cases, even though the same primer sequences are being aligned. To resolve this issue, we internally adjust the specified expect value cutoff for the template case using the length of the artificial template from the primer-only case as a guide. This ensures that the BLAST results are equivalent between submitting a template and submitting primers only.

QPCR primers that anneal poorly or that anneal to more than one sequence during amplification can significantly impact the quality and reliability of your results. Methylation Specific PCR (MSP) relies on amplification to assess the methylation status at specific CpG sites after bisulfite conversion. Success with this system depends on the differential amplification of the template using methylated (M) and non-methylated (U) primer sets. While most of the considerations for MSP primer design are identical for those for bisulfite PCR, CpG sites within the primers are treated differently. Bisulfite PCR examines the methylation status of all the CpG sites present in a specific region, while MSP assesses the methylation at a single CpG site complementary to the 3’ end of the primer sequence.

The results page reports the specificity of the generated primers, a graphic summary of primer pairs in relation to the PCR template and certain features such as exons, as well as detailed information on each primer pair. It will only show target-specific primers if found; otherwise, it will report all primers. In all cases, the actual targets will be listed along with detailed alignments between primers and targets.