Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions

Abstract

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced 'quantitative' C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.

Figure 1

A continuous sequence of DNA representing the bisulfite-modified COMT promoter region amplified as a template for the SNaPshot multiplexing experiments. Only the top strand of DNA is depicted along with the positions of all SNaPshot primers used. Polymorphic target sites created by bisulfite modifications of M-HpaII and M-HhaI methylated amplicons are marked in bold capital letters. Primers overlapping M-HhaI sites (bottom right) were designed with T at the overlapping CpG positions. Forward SNaPshot primers are boxed above the sequence while reverse SNaPshot primers are boxed in red below the sequence. The non-binding GACT repeat tails (placed on the 3′ end of some primers) are denoted by a number, the purpose of which is to vary the primer length in order to distinguish them in the ABI 3100 Genetic Analyzer.

Figure 2

Oligonucleotide templates and complementary primers were synthesized to test the effects of C/T and G/A polymorphisms upstream of the target nucleotide at positions −2, −5, −10, −15 and −18 bp. Sequences are depicted for C/T and G/A polymorphisms at positions −2, −10 and −18; however, primers and templates were also tested for positions −5 and −10. Templates are named for the complementary strand to which the SNaPshot primer binds, the two nucleotides representing the polymorphic position (signified by the number) and the target nucleotide, respectively. SNaPshot primers are named according to the nucleotide complementary to the upstream polymorphism.

Figure 3

A list of oligonucleotide templates synthesized to contain between 1 and 4 polymorphic positions in the SNaPshot primer binding region and respective primers containing degenerative bases at positions corresponding to those polymorphisms. Percentages of nucleotides synthesized into the templates are depicted while SNaPshot primers were designed with a 50%:50% proportion of C/T at all polymorphic positions.

Figure 4

SNaPshot primers for the galectin1 (A) and humanin (B) genes that were identified as being differentially methylated between placenta and brain tissue.

Figure 5

A graph of the average methylation values reported by nine primers interrogating control templates created to contain only C or T at the CpG islands of interest. Each dilution series was tested in triplicate for each primer so that each data point is an average of 27 experiments. Templates with all CpG islands of interest containing C were diluted and mixed in increments of 5% with templates containing T at all CpG being interrogated to test the ability of the primers to accurately measure the amount of methylation.

Figure 6

Data output from the ABI Avante 3100 combining 60% bisulfite-treated M-HpaII methylated template and 40% unmethylated template. The peak heights show 60% C to 40% T signal for those peaks methylated with M-HpaII (peak pairs 1, 2 and 4). Peaks 3, 5 and 6, representing M-HhaI sites, show no C signal and hence no methylation. Peak order is determined by primer size.

Figure 7

A graphical representation of the percentage that a mismatch in the primer binding region at various positions upstream from the target CG can affect the reading. There is a correlation between the proximity of a primer mismatch to the target and the degree to which the resulting ^metC/C reading will be inaccurate. Zero baseline represents 0% biasing effect.

Figure 8

(A) Data points produced by 25% increments of G_target/A_target templates while varying the percentage of polymorphic G/A 2 bp upstream from the target nucleotide. (B) Data points produced by 25% increments G_target/A_target templates while varying the percentage of polymorphic G/A 5 bp upstream from the target nucleotide. Results for each data point in the −2 (A) and −5 (B) permutations represent an average of 10 experiments. (C) Data points produced by diluting the G_target/A_target 25% increments while varying the percentage of polymorphic nucleotides in the amounts shown to the right.

Figure 9

Graphical representation of methylation profiles quantified by sequencing of at least 12 clones of bisulfite-modified genomic DNA and SNaPshot for six CpG dinucleotide positions in bisulfite-modified DNA amplified from brain tissue (A) and placenta tissue (B).

Figure 10

(A) SNaPshot results on bisulfite-modified DNA from brain interrogated with primers gal1, gal2 and gal3. (B) The peak pattern identified with gal1 SnaPshot primer only. (C) A depiction of how the proportion of multiple peaks in the scenario of a single upstream polymorphism were indicative of the methylation profile of the upstream CpG (Y position) measured by gal1 and verified by sequencing of bisulfite-modified genomic DNA. (D) SNaPshot peaks resultant from an interrogation of the gal1 upstream CpG (Y) using primer sequence: 5′-TTGGGGGTTATTGGGGG-3′.