PCR Incorporation of Modified dNTPs: The Substrate Properties of Biotinylated dNTPs

Abstract

Modified dNTPs are a convenient tool for the enzymatic introduction of functional groups into a nucleic acid target of interest. One major benefit of enzymatic functionalization is uniform distribution of modifications across a long target sequence. In comparison, using modified oligonucleotides is an appropriate choice when shorter sequences modified in discrete locations are desired. Nick translation and random labeling are the most commonly used methods for modified dNTP incorporation. These methods are generally applied to complex nucleic acid mixtures (1,2). When a specific primer pair is used in combination with DNA polymerase, a functionalized double stranded DNA copy of a defined region is generated. Although modified dNTPs can be employed in a number of different enzymatic protocols, the position and type of attachment have a strong influence on DNA polymerase efficiency. Extensive investigation has revealed that modifications to the major groove of the nucleobase allow for the best incorporation efficiency with the 5-position of pyrimidines and the 7-position of purines being the optimal site (1). The flexibility of the linker arm attaching the modification can also influence nucleotide utilization, with rigid, linear linkers providing the strongest dNTP substrate properties. Linker arm length also plays a role in modified dNTP incorporation. Modified dNTPs with shorter linker arms (i.e., biotin4-dUTP) are better substrates than nucleotides with longer linker arms (i.e., biotin-11-dUTP or biotin-14-dUTP) (2). With the knowledge of linker position and length, functional groups can be introduced for each of the four dNTPs. However, often only one of the four modified dNTPs is offered commercially, with 5-substituted dUTP analogs being the most readily available. There are a number of considerations for efficient amplicon formation when employing modified dNTPs. While Taq DNA polymerase is traditionally used, in some instances members of the B-family of polymerases provide greater PCR efficiency. Furthermore, complete substitution of a modified dNTP for its natural counterpart causes inhibition of PCR, resulting in low and often undetectable product formation. This is due to the compounding effect of a lower efficiency of nucleotide incorporation over multiple cycles. In order to achieve a high degree of functionalization without compromising yield, the percentage of substitution of a modified dNTP for its natural counterpart needs to be experimentally determined. Herein we will describe an approach for modified dNTP incorporation in PCR, using biotinylated dNTPs. The goal is to label the resultant amplicon at high density with biotin using 5-modified biotin-16-aminoallyl dUTP and dCTP.