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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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Related in: MedlinePlus

Position-dependent nucleotide frequency maps (sequence logos) (A) low P90 (fast amplification) (B) large positive Diff (good temporal separation between specific and non-specific amplification) (C) high P90 (slow amplification), and (D) large negative Diff (poor temporal separation between specific and non-specific amplification). A–D were generated by an online graphical sequence representation tool (54,55) using the template sequences which were used to generate PWM. At each position, the size of the overall stack indicates the conservation of that position, while the size of each symbol represents the frequency of that nucleic acid at that position. (E) Shannon entropy (56) for each variable position in Class I templates (blue dashed line), Class II templates (red dotted line) and Class I and Class II combined (orange solid line).
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gks230-F9: Position-dependent nucleotide frequency maps (sequence logos) (A) low P90 (fast amplification) (B) large positive Diff (good temporal separation between specific and non-specific amplification) (C) high P90 (slow amplification), and (D) large negative Diff (poor temporal separation between specific and non-specific amplification). A–D were generated by an online graphical sequence representation tool (54,55) using the template sequences which were used to generate PWM. At each position, the size of the overall stack indicates the conservation of that position, while the size of each symbol represents the frequency of that nucleic acid at that position. (E) Shannon entropy (56) for each variable position in Class I templates (blue dashed line), Class II templates (red dotted line) and Class I and Class II combined (orange solid line).

Mentions: In addition to facilitating assay design, another motivation for this study was to better understand which sequence motifs give rise to efficient specific EXPAR amplification in the presence of trigger, and which motifs facilitate non-specific background amplification. Using the PWM approach, we have determined position-dependent nucleotide frequency maps (54,55) for templates that exhibit fast versus slow amplification (Figure 9A and C) and for templates with good versus poor separation between specific and non-specific amplification (Figure 9B and D). We further calculated the entropy (randomness) (56) of each variable position within Class I templates only, Class II templates only, and Class I and II templates combined (Figure 9E).Figure 9.


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)

Position-dependent nucleotide frequency maps (sequence logos) (A) low P90 (fast amplification) (B) large positive Diff (good temporal separation between specific and non-specific amplification) (C) high P90 (slow amplification), and (D) large negative Diff (poor temporal separation between specific and non-specific amplification). A–D were generated by an online graphical sequence representation tool (54,55) using the template sequences which were used to generate PWM. At each position, the size of the overall stack indicates the conservation of that position, while the size of each symbol represents the frequency of that nucleic acid at that position. (E) Shannon entropy (56) for each variable position in Class I templates (blue dashed line), Class II templates (red dotted line) and Class I and Class II combined (orange solid line).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks230-F9: Position-dependent nucleotide frequency maps (sequence logos) (A) low P90 (fast amplification) (B) large positive Diff (good temporal separation between specific and non-specific amplification) (C) high P90 (slow amplification), and (D) large negative Diff (poor temporal separation between specific and non-specific amplification). A–D were generated by an online graphical sequence representation tool (54,55) using the template sequences which were used to generate PWM. At each position, the size of the overall stack indicates the conservation of that position, while the size of each symbol represents the frequency of that nucleic acid at that position. (E) Shannon entropy (56) for each variable position in Class I templates (blue dashed line), Class II templates (red dotted line) and Class I and Class II combined (orange solid line).
Mentions: In addition to facilitating assay design, another motivation for this study was to better understand which sequence motifs give rise to efficient specific EXPAR amplification in the presence of trigger, and which motifs facilitate non-specific background amplification. Using the PWM approach, we have determined position-dependent nucleotide frequency maps (54,55) for templates that exhibit fast versus slow amplification (Figure 9A and C) and for templates with good versus poor separation between specific and non-specific amplification (Figure 9B and D). We further calculated the entropy (randomness) (56) of each variable position within Class I templates only, Class II templates only, and Class I and II templates combined (Figure 9E).Figure 9.

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
Related in: MedlinePlus