Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

Abstract

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Figure 1

Mechanism of the Invader assay (A) and the Invader squared assay (B).

Figure 2

SNP genotyping by the Invader squared assay and MALDI-TOF MS.

Figure 3

Four representative genotyping results. The two possible nucleotides at the polymorphic position are given in brackets next to the name of each SNP analyzed. The expected signal molecule deprotonated, negative singly charged ion ([M − H]⁻) m/z for each of the possible two alleles is also given, along with (i) the MALDI-TOF MS analysis result and (ii) the relevant portion of the four-color sequencing trace, where the polymorphic nucleotide position is given in brackets.