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-nitro-dGTP, 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 and/or 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.

Fig 1.

The chemical structures of modified nucleotides used in ICCC genotyping. The modified groups are highlighted.

Fig 2.

Schematic of the ICCC genotyping strategy. A* represents a chemically modified analog of A.

Fig 3.

Nondenaturing PAGE (10%) analysis of PCR products using modified nucleotides. (A) Gel stained with 1:1 mixture of ethidium bromide and SYBR green I dyes. (B) An autoradiogram of the gel shown in A.

Fig 4.

PAGE analysis of cleavage products from PCR samples shown in Fig. 3. (A) An autoradiogram of cleavage products from pyrrolidine treatment alone. (B) An autoradiogram of cleavage products from chemical reactions with KMnO₄ followed by pyrrolidine. (C) Profile presentation of B. ▾ points to genotype calling peaks.

Fig 5.

MALDI-TOF mass spectra of chemical cleavage products from treating 12 PCR products listed in Table 1 with KMnO₄ followed by 3-pyrrolidinol. Genotype calling fragments are indicated by shaded MWs.

Fig 6.

Representative, high-throughput cytochromes P450 2C9 (A) and 2D6 (B) genotyping results for samples with different genotypes. ▾ points to allele-diagnostic mass peaks. C shows the sequences of the PCR amplicons and cleavage fragments (see supporting information on the PNAS web site for additional sequence and mass information).