Genomic DNA can participate in the creation of both the desired amplicon and the primer dimerization artifact. Instead, this mechanism requires that sites for P1 and P2 are close to each other. DNA Software Phone: 1 Skip to content. An Alternative Mechanism for Primer Dimer Artifacts There are some additional observations that provide clues for an alternative mechanism for primer dimerization. Generally, homodimers i.
Primer dimers increase markedly when heterologous genomic DNA is added. Primer dimers are most often observed when one or both of the primers bind inefficiently to the target DNA e. Arrow heads indicate the direction of DNA synthesis. The nucleotide sequences of the synthetic oligonucleotides are shown in Table 1. Primers for the PD synthesis reactions comprise two groups and are aligned in Figure 2.
Applied Biosystems. PCRs were performed using a Hybaid Omnigene cycler. A Alignment of the primers comprising group 1. B Alignment of the primers comprising group 2. Thermal cycling using a P.
Forward and reverse sequencing reactions were carried out using 0. Dye terminator cycle sequencing was performed using a Taq dye terminator kit P. Applied Biosystems by a P. Applied Biosystems automated DNA sequencer. A contig from the unedited forward and reverse sequences for each clone was generated using the SeqMan software of DNAStar. Tag sequences were designed to exhibit the following characteristics: i they should have no known genomic target themselves; ii they should have high T m s; iii they should not be prone to PD formation by themselves or with other primers in the reactions; and iv they should have no stable secondary structure.
Oligonucleotides 13mers were designed incorporating the least frequent neighbouring pairs of bases 22 and then examined for their absence in known human genomic sequences in the GenBank database. They were then combined to generate candidate 26mers which were examined for self- and human genomic DNA complementarity.
Tag sequences are shown in Table 1. All DNA samples were prepared as described previously 3. Thermal cycling was performed in 0.
Applied Biosystems Thermal Cycler. All procedures generally accepted for avoiding PCR carry-over contamination were employed After PCR, reaction mixes for each PD were pooled and the whole of each sample was electrophoresed on a 1.
For each PD there was an equivalent no PD negative control. Primers, template and polymerase additions were as shown in Table 2. The excitation wavelength was nm and the emission filter was nm. DNA sequence alignments of cloned PDs.
Contigs derived from the forward and reverse sequencing of individual cloned PDs aligned against the primers from which they were produced. Rev comp. Nucleotides shown in lower case appear to be deleted from the primers during PD formation. The accumulation of PDs when a primer from either group 1 or group 2 was combined with another from the same group Fig.
PD synthesis was pronounced when primers from each group were combined. Any one primer in isolation would not give rise to detectable PDs as shown previously R. Ferrie, unpublished. These observations were upheld irrespective of any complementarity between primers Table 3.
An analysis of alignments of cloned PD sequences and the primers from which they were formed is shown in Figure 3. PD 2 was for use with separate Tag sequences. Homo-Tailed PD1 was only visible on gel analysis when 10 10 target molecules were present in the reaction. In contrast, the Hetero-Tailed PD 2 was efficiently amplified to produce a visible band on a gel when as few as 10 molecules of target were present in the reaction. The Homo-Tag format therefore suppressed PD formation by a factor of 10 9.
By increasing the annealing temperature of the PCR cycles above the T m of the Tail sequence, PD1 was re-amplified when fewer 10 6 template molecules were present in the reaction data not shown. PD reamplification. Control and ARMS allele-specific amplicons are shown. Genomic untailed primers gave rise to specific amplicon in the presence of genomic DNA only and to PDs either in the presence or absence of genomic DNA.
Similar results were obtained using tailed equivalents to the genomic primers. Table 4 shows mean fluorescence readings from the reactions that contained Taq DNA polymerase. Only when the appropriate allele was present was the respective amplicon produced; furthermore, there was no visible PD accumulation Fig. PDs are routinely observed in PCRs. They are not derived from template DNA and they can complicate experimental analysis. General concepts for PCR primer design.
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The authors wish to acknowledge Dr. Meredith McNeil and Dr. Andrew D. You can also search for this author in PubMed Google Scholar. Correspondence to Darren Korbie or Matt Trau.
Reprints and Permissions. Johnston, A. Sci Rep 9, Download citation. Received : 13 February Accepted : 23 November Published : 18 January Anyone you share the following link with will be able to read this content:.
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Download PDF. Subjects Epigenetics Software. Introduction Primer design software seeks to maximize product yield and minimize off-target amplification, and a key component of this is the prevention of the primer-primer interaction artefacts known as primer-dimers. Results and Discussion Primer-dimer prediction To begin development on our dimer prediction algorithm PrimerDimer, a set of primer-dimer artefacts were first sequenced to assess the types of primer-primer interactions that cause dimer formation Supplementary Fig.
Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure Material and Methods Assessment of primer-dimer prediction programs To determine the accuracy of different dimer algorithms in predicting the formation of primer-dimers, the dimer score i.
PerlPrimer Used PerlPrimer version 1. Oligo 7 Used Oligo 7 version 7. OligoAnalyzer Used OligoAnalyzer version 3. Primer3 Used Primer3 version 2. References 1. Article Google Scholar Google Scholar Download references. Acknowledgements The authors wish to acknowledge Dr. Author information Author notes Andrew D. Johnston and Jennifer Lu contributed equally. Johnston View author publications. View author publications. Ethics declarations Competing Interests The authors declare no competing interests.
Electronic supplementary material. Supplementary Info. About this article. Cite this article Johnston, A. Copy to clipboard. Song , Wenjia Qu , Gail P.
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