Document Detail

Conversion strategy using an expanded genetic alphabet to assay nucleic acids.
MedLine Citation:
PMID:  23541235     Owner:  NLM     Status:  MEDLINE    
Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.
Zunyi Yang; Michael Durante; Lyudmyla G Glushakova; Nidhi Sharma; Nicole A Leal; Kevin M Bradley; Fei Chen; Steven A Benner
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-04-17
Journal Detail:
Title:  Analytical chemistry     Volume:  85     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2013 May 
Date Detail:
Created Date:  2013-06-28     Completed Date:  2013-12-05     Revised Date:  2014-05-08    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4705-12     Citation Subset:  IM    
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MeSH Terms
Nucleic Acid Conformation
Nucleic Acids / chemistry,  genetics*
Polymerase Chain Reaction
Sequence Analysis, DNA*
Sequence Analysis, RNA*
Grant Support
Reg. No./Substance:
0/Nucleic Acids

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