Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF

Abstract

The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.

Figure 1

Scheme of P-N-bond fragmentation assay. The experimental protocol is described in Methods.

Figure 2

Fragmentation analysis of an APOB target by using an optimized cleavage protocol. (A) Sequence of a 265-bp APOB amplicon and the list of fragments resulting from a P-N dT-specific cleavage of an extension product. Locations of PCR and extension primers are labeled. Potential cleavage sites (dT) are indicated in bold. (B) Experimentally obtained fragmentation mass spectrum of a 265-bp product. Only measured peak m/z values are shown.

Figure 2

Fragmentation analysis of an APOB target by using an optimized cleavage protocol. (A) Sequence of a 265-bp APOB amplicon and the list of fragments resulting from a P-N dT-specific cleavage of an extension product. Locations of PCR and extension primers are labeled. Potential cleavage sites (dT) are indicated in bold. (B) Experimentally obtained fragmentation mass spectrum of a 265-bp product. Only measured peak m/z values are shown.

Figure 3

Identification of a single base insertion by fragmentation. A CETP target was analyzed as described in the text. (A, B) Mass spectra of P-N dTTP and P-N dCTP incorporation reactions, respectively. (C) Scheme showing cleavage sites in both reactions revealing a single dG insertion.

Figure 3

Identification of a single base insertion by fragmentation. A CETP target was analyzed as described in the text. (A, B) Mass spectra of P-N dTTP and P-N dCTP incorporation reactions, respectively. (C) Scheme showing cleavage sites in both reactions revealing a single dG insertion.

Figure 4

Automated fragmentation analysis of NFKBIA. (A) Analyzed fragment of the NFKBIA gene. PCR and extension primers, three interrogated polymorphisms, and a stop codon (boxed) are shown. (B-E) Representative mass spectra of observed genotypes. Note that the genotype exemplified in E was measured in a single sample only.

Figure 5

Incomplete (partial) P-N-bond cleavage. APOB extension product (265 bp) was generated as in Figure 2B with the exception that 10 μM natural dTTP was incorporated together with 5 mM P-N dTTP. Note the increased total number of fragmentation peaks compared with Figure 2B. Representative incomplete cleavage peaks are labeled by a crossed circle.