A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides

Abstract

Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitrodGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and͞or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.

7-Deazapurine (Pyrrolo[2,3-d]pyrimidine) 2-Deoxyribonucleosides: Syntheses and Transformations

“…Nitration of 47 (HNO 3 /H 2 SO 4 48) followed by deprotection (NaOMe/MeOH) gave 7-deaza-2'-deoxy-7-nitroadenosine (14g). 7-Deaza-7-nitropurine nucleosides, such as 14g and 7-deaza-2'deoxy-7-nitroguanosine (12g) have been utilized for DNA genotyp-ing (Incorporation and Complete Chemical Cleavage, ICCC) [80] and a footprinting technique (Template Directed Interference, TDI) [81]. The synthesis of 12g was performed as described in section 3.2.4 (Scheme 29).…”

Advances in the Synthesis of 7-Deazapurine - Pyrrolo[2,3-d]pyrimidine 2-Deoxyribonucleosides Including D- and L-Enantiomers, Fluoro Derivatives and 2,3-Dideoxyribonucleosides

“…Nitration of 46 (HNO 3 /H 2 SO 4 , 47) followed by deprotection (NaOMe/MeOH) gave 7-deaza-2'-deoxy-7-nitroadenosine (13g). 7-Deaza-7-nitropurines, such as 13g and 7-deaza-2'-deoxy-7-nitroguanosine (11g) have been utilized for DNA genotyping (Incorporation and Complete Chemical Cleavage, ICCC) [66] and a footprinting technique (Template Directed Interference, TDI) [67]. The synthesis of 11g was performed as described in section 3.2.4 (Scheme 34).…”

Genotyping single nucleotide polymorphisms by mass spectrometry

“…The incorporation and complete chemical-cleavage assay (ICCC) relies on the incorporation of one chemically labile nucleotide such as 7-deaza-7-nitro-deoxypurines and 5-hydroxy-deoxypyrimidines during PCR (Wolfe et al, 2002). Amplicons ($70 bases) are subsequently fragmented in a chemical reaction by specific and quantitative cleavage at the positions of the modified nucleotides.…”