Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

Abstract

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Figure 1

Principle of allele-specific primer elongation on microarrays. Single-stranded DNA targets serve as templates in a Taq polymerase-catalyzed elongation reaction of immobilized oligonucleotide primers. The match and mismatch primers differ at their free 3′-end by a variable base, which is discriminated by the enzyme. Elongation and thereby incorporation of Cy3-dUTP takes place in a template-dependent manner.

Figure 2

Mismatch detection using synthetic oligonucleotides as targets. Four primer elongation reactions were performed with a 50mer oligonucleotide carrying a central single nucleotide exchange, in each reaction. Oligonucleotide primers spotted as duplicates on the microarray, consisted of a 5′(T)₁₅ spacer sequence linked to a 25 bp specific sequence complementary to the 25 nt at the 3′-end of the targets and carried one of the four bases at their free 3′-ends. Positions of the primers with the respective variable base are indicated above.

Figure 3

False positive signals under different experimental conditions. Primer elongation reactions were performed under standard conditions (see Materials and Methods) without a target on differently coated slides. (A) 1%, (B) 2%, (C) 3% aminopropyl-trimethoxysilane and (D) aminosilane slides from Perkin Elmer. Prominent false positive signals are denoted by arrows. Possible primer–dimer structures of five of these six sequences are shown below. The false positive signal at position 2 cannot be explained by the formation of primer–dimers or hairpin loops. Varying parameters of the cycling protocol influence the intensity of false positive signals: (E) standard conditions, (F) increased cycle number (25 instead of 15), (G) decreased annealing temperature (48 instead of 56°C), (H) prolonged annealing time (60 instead of 30 s) and (I) prolonged elongation time (30 instead of 15 s).

Figure 4

Detection of nine SNPs in a 426 bp sequence using differently generated single-stranded DNA targets. (A) Illustration of the relative position and kind of SNPs within the template with primers indicated by an arrow. (B) Result of a primer elongation reaction with asymmetric PCR products as targets, which were generated in a reaction with a single PCR primer as indicated by a bold arrow in (A). (C) SNP detection using asymmetric PCR products generated with all six primers in a multiplex reaction. (D) An exonuclease-treated PTO-modified PCR product spanning the complete 426 bp sequence was used in a primer elongation reaction.

Figure 5

Primer elongation reaction on a microarray carrying paired oligonucleotide primers corresponding to 48 different mitochondrial SNPs. Asymmetric PCR products generated in three multiplexed reactions with 13, 15 and 20 primers were used as targets. Match (marked in gray) and mismatch bases of the immobilized primers as well as match to mismatch ratios of the fluorescent signals are listed in the table according to the position on the microarray.

Figure 6

Detection of 46 mitochondrial SNPs contained in five large targets generated by exonuclease-treatment of the respective PTO-modified PCR products (A, B, C1, C2 and D), which span the mitochondrial genome. Particular SNPs are found in overlapping targets as depicted by identical numeration. Paired match and mismatch oligonucleotide primers are arranged in alternate rows with the exception of a few pairs, as indicated by oblique arrows.