NDLI: Use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction.

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Use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction.

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Use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Kutyavin, Igor V.
Description	Country affiliation: United States Author Affiliation: Kutyavin IV ( Perpetual Genomics, 18943 203rd Ave. NE, Woodinville, WA 98077, USA. perpetualgenomics@comcast.net)
Abstract	The addition of relatively short flap sequence at the 5'-end of one of the polymerase chain reaction (PCR) primers considerably improves performance of real-time assays based on 5'-nuclease activity. This new technology, called Snake, was shown to supersede the conventional methods like TaqMan, Molecular Beacons, and Scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. The present article describes a number of reaction conditions and methods that allow further improvement of the assay performance. One of the identified approaches is the use of duplex-destabilizing modifications such as deoxyinosine and deoxyuridine in the design of the Snake primers. This approach was shown to solve the most serious problem associated with the antisense amplicon folding and cleavage. As a result, the method permits the use of relatively long-in this study-14-mer flap sequences. Investigation also revealed that only the 5'-segment of the flap requires the deoxyinosine/deoxyuridine destabilization, whereas the 3'-segment is preferably left unmodified or even stabilized using 2-amino deoxyadenosine d(2-amA) and 5-propynyl deoxyuridine d(5-PrU) modifications. The base-modification technique is especially effective when applied in combination with asymmetric three-step PCR. The most valuable discovery of the present study is the effective application of modified deoxynucleoside 5'-triphosphates d(2-amA)TP and d(5-PrU)TP in Snake PCR. This method made possible the use of very short 6-8-mer 5'-flap sequences in Snake primers.
File Format	HTM / HTML
ISSN	1540658X
e-ISSN	15578127
DOI	10.1089/adt.2010.0303
Journal	ASSAY and Drug Development Technologies
Issue Number	1
Volume Number	9
Language	English
Publisher	Mary Ann Liebert, Inc.
Publisher Date	2011-02-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Analytical Discipline Pharmacology Discipline Chemistry Techniques Dna Primers Genetics Nucleic Acids Nucleotides Polymerase Chain Reaction Sequence Analysis, Dna Molecular Sequence Data Sensitivity And Specificity Research Support, N.i.h., Extramural
Content Type	Text
Resource Type	Article
Subject	Drug Discovery Molecular Medicine

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Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

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Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

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