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INTEGRATEDDNATECHNOLOGIESModified BasesPRODUCTSMODIFICATIONSdeoxyInosine MW: 314.2Historically, the first universal base employed was 2’-deoxyInosine (dI). DeoxyInosine is a naturally occurring base that, whilenot truly universal, is less destabilizing than mismatches involving the four standard bases. Hydrogen bond interactionsbetween dI and dA, dG, dC and dT are weak and unequal, with the result that some base-pairing bias does exist with dI:dC >dI:dA > dI:dG > dI:dT. When present in a DNA template, deoxyInosine preferentially directs incorporation of dC in the growingnascent strand by DNA polymerase.5’ INT. 3’a a a'5OHNONONN5' deoxyInosineLocked Nucleic Acids (LNAs)OO P O -O3'LNA A MW: 341.2 LNA G MW: 357.2LNA C MW: 331.2 LNA T MW: 332.2LNA bases have a modification to the ribose backbone that locks the base in the 3’-endo position, which favors RNA A-typehelix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertionscan be placed in an oligo at any position. Applications have been described ranging from antisense oligos to hybridizationprobes to SNP detection and allele -specific PCR. Due to the large increase in Tm conferred by LNAs, they also can causean increase in primer dimer formation as well as self-hairpin formation. We recommend limiting the number of LNAsincorporated into a single oligo to 10 bases or less 1,2 . LNA bases are indicated using the + symbol (+A for example) in the IDTordering system.5’ INT. 3’a a a'5OOOO P O -OBaseReferences:O3'1. Design considerations and effects of LNA in PCR primers. Latorra, D., Arar, K., and Hurley, J.M., Mol. Cell. Probes., 17:253-9 (2003).2. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimeradesign. Braasch, D.A., Liu, Y., and Corey, D.R., Nucleic Acids Res., 30:5160-7 (2002).5-Nitroindole MW: 340.25-Nitroindole is the best universal base currently available. It does not favor any particular base-pairing (i.e., it does notsupport base-specific hydrogen bond formation), but does contribute to duplex stability through base-stacking interactions.Therefore, it is not as destabilizing to the duplex as mismatches between the standard bases. 5-Nitroindole directs randomincorporation of any base when used as a template for DNA polymerase and partially blocks enzyme processivity 1 .5’ INT. 3’a a —O 2NReference:1. The applications of universal DNA base analogues. Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001).'5OONOO P O -O3'©2008 Integrated DNA Technologies 78 www.idtdna.com