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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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Example template sequence #1 (11) consisting of (A) the trigger reverse complement X′, which is replicated at positions 1–10 and 21–30, (B) the nicking enzyme post-cut site, (C) the reverse complement of the nicking enzyme recognition site, (D) an additional T at the end of the trigger-binding site.
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gks230-F2: Example template sequence #1 (11) consisting of (A) the trigger reverse complement X′, which is replicated at positions 1–10 and 21–30, (B) the nicking enzyme post-cut site, (C) the reverse complement of the nicking enzyme recognition site, (D) an additional T at the end of the trigger-binding site.

Mentions: Out of the 384 template sequences used in this study (Supplementary Table S1), sequences #1–64 had previously been characterized in our laboratory, whereas sequences #65–384 were newly designed. Design of sequences #2–11 was based on sequence #1 (Figure 2) described in an earlier publication (11) with a single nucleotide base change each in the trigger complement positions 1–10. For all remaining 373 sequences, we randomly assigned A, T, C, or G with equal probability to each of the 14 variable positions that make up the trigger complement and nicking enzyme post-cut site (Figure 2; positions 1–14, with 1–10 replicated at 21–30). An additional ‘T’ was added to each template at the 3′ end of the nicking enzyme recognition site (position 20), because we earlier hypothesized that the polymerase may append an A to newly generated triggers through non-templated adenylation. Although we found this not to be the case under the current EXPAR conditions, based on mass spectrometric analysis of the generated trigger, we kept the extra T in the template sequences for consistency.Figure 2.


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)

Example template sequence #1 (11) consisting of (A) the trigger reverse complement X′, which is replicated at positions 1–10 and 21–30, (B) the nicking enzyme post-cut site, (C) the reverse complement of the nicking enzyme recognition site, (D) an additional T at the end of the trigger-binding site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks230-F2: Example template sequence #1 (11) consisting of (A) the trigger reverse complement X′, which is replicated at positions 1–10 and 21–30, (B) the nicking enzyme post-cut site, (C) the reverse complement of the nicking enzyme recognition site, (D) an additional T at the end of the trigger-binding site.
Mentions: Out of the 384 template sequences used in this study (Supplementary Table S1), sequences #1–64 had previously been characterized in our laboratory, whereas sequences #65–384 were newly designed. Design of sequences #2–11 was based on sequence #1 (Figure 2) described in an earlier publication (11) with a single nucleotide base change each in the trigger complement positions 1–10. For all remaining 373 sequences, we randomly assigned A, T, C, or G with equal probability to each of the 14 variable positions that make up the trigger complement and nicking enzyme post-cut site (Figure 2; positions 1–14, with 1–10 replicated at 21–30). An additional ‘T’ was added to each template at the 3′ end of the nicking enzyme recognition site (position 20), because we earlier hypothesized that the polymerase may append an A to newly generated triggers through non-templated adenylation. Although we found this not to be the case under the current EXPAR conditions, based on mass spectrometric analysis of the generated trigger, we kept the extra T in the template sequences for consistency.Figure 2.

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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