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Sequence dependence of isothermal DNA amplification via EXPAR.

Qian J, Ferguson TM, Shinde DN, Ramírez-Borrero AJ, Hintze A, Adami C, Niemz A - Nucleic Acids Res. (2012)

Bottom Line: Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates.Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers.Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

View Article: PubMed Central - PubMed

Affiliation: Keck Graduate Institute, Claremont, 535 Watson Drive, Claremont, CA 91711, USA.

ABSTRACT
Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67-70% sensitivity and 77-80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

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Results of data analysis and classification: mean values and standard errors of (A) Diff plotted versus P90 and (B) N10 plotted versus P90 for 307 template sequences that were classified into 102 Class I templates (good performers, blue diamond), 102 Class II templates (poor performers, red circle) and 103 Class III templates (intermediate performance, orange triangle).
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gks230-F5: Results of data analysis and classification: mean values and standard errors of (A) Diff plotted versus P90 and (B) N10 plotted versus P90 for 307 template sequences that were classified into 102 Class I templates (good performers, blue diamond), 102 Class II templates (poor performers, red circle) and 103 Class III templates (intermediate performance, orange triangle).

Mentions: Following the exclusion criteria described in the ‘Materials and methods’ section, we obtained suitable data for 307 template sequences, which were used for further analysis. The performance of these 307 template sequences (Figure 5) varied significantly. In the graph of Diff versus P90 (Figure 5A), well-performing templates appear at low P90 and positive Diff values, while poorly performing templates appear at high P90 and negative Diff values. In poorly performing templates with minimal or no separation between positive and negative amplification curves, the Diff value can become negative, since Diff measures the separation between P90 and N10, not between the inflection points of the positive and negative amplification reactions. For example, the sequence shown in Figure 3B has a Diff of negative 13 min. Conversely, for template sequences that show good temporal separation between specific and non-specific amplification, Diff is smaller than the actual separation between the inflection points of the positive and negative curves. Using Diff for performance characterization also penalizes templates with shallow amplification kinetics. Figure 5A and B contains the same information, but in a different graphical representation. In the graph of N10 versus P90 (Figure 5B), well-performing templates appear above the diagonal at lower P90 values, while poorly performing templates appear below the diagonal.Figure 5.


Sequence dependence of isothermal DNA amplification via EXPAR.

Qian J, Ferguson TM, Shinde DN, Ramírez-Borrero AJ, Hintze A, Adami C, Niemz A - Nucleic Acids Res. (2012)

Results of data analysis and classification: mean values and standard errors of (A) Diff plotted versus P90 and (B) N10 plotted versus P90 for 307 template sequences that were classified into 102 Class I templates (good performers, blue diamond), 102 Class II templates (poor performers, red circle) and 103 Class III templates (intermediate performance, orange triangle).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367216&req=5

gks230-F5: Results of data analysis and classification: mean values and standard errors of (A) Diff plotted versus P90 and (B) N10 plotted versus P90 for 307 template sequences that were classified into 102 Class I templates (good performers, blue diamond), 102 Class II templates (poor performers, red circle) and 103 Class III templates (intermediate performance, orange triangle).
Mentions: Following the exclusion criteria described in the ‘Materials and methods’ section, we obtained suitable data for 307 template sequences, which were used for further analysis. The performance of these 307 template sequences (Figure 5) varied significantly. In the graph of Diff versus P90 (Figure 5A), well-performing templates appear at low P90 and positive Diff values, while poorly performing templates appear at high P90 and negative Diff values. In poorly performing templates with minimal or no separation between positive and negative amplification curves, the Diff value can become negative, since Diff measures the separation between P90 and N10, not between the inflection points of the positive and negative amplification reactions. For example, the sequence shown in Figure 3B has a Diff of negative 13 min. Conversely, for template sequences that show good temporal separation between specific and non-specific amplification, Diff is smaller than the actual separation between the inflection points of the positive and negative curves. Using Diff for performance characterization also penalizes templates with shallow amplification kinetics. Figure 5A and B contains the same information, but in a different graphical representation. In the graph of N10 versus P90 (Figure 5B), well-performing templates appear above the diagonal at lower P90 values, while poorly performing templates appear below the diagonal.Figure 5.

Bottom Line: Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates.Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers.Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

View Article: PubMed Central - PubMed

Affiliation: Keck Graduate Institute, Claremont, 535 Watson Drive, Claremont, CA 91711, USA.

ABSTRACT
Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67-70% sensitivity and 77-80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions.

Show MeSH