2, Christian Albrecht University, Kiel, Germany
In Pyrosequencing™, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3′ end to itself. In the resulting structure, the 3′ end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3′ end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3′ end and (ii) the extension of the template's 3′ end with a ddNMP. In contrast to unprotected 3′ ends of ssDNA templates, causing inconsistent results, we show that protecting the 3′ end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.
Pyrosequencing™ is a high-throughput method for sequencing and genotyping short DNA fragments. In the sequencing reaction, pyrophosphates that are split off from the deoxynucleoside triphosphates during PCR are quantified by a cascade of enzymatic reactions. At the cascade's end, the indicator reaction catalyzed by luciferase results in detectable light that correlates to the nucleotides initially incorporated. Compared to other genotyping methods, Pyrosequencing is attractive because it allows both single nucleotide polymorphism (SNP) detection within its sequence context and calculation of the amounts of variant alleles in a single experiment. Pyrosequencing is therefore especially useful for estimating allele frequencies with pooled genomic DNA and for detecting allelic imbalances in cDNA samples.
Pyrosequencing is performed isothermically at 28°C with single-stranded DNA (ssDNA) as a template. These conditions favor the formation of secondary structures within the template and may even result in self-priming of the ssDNA 3′ ends, which generates artificial signals and ghost peaks, even in the absence of the sequencing primer ((Figure 1)A) (1,2,3).Figure 1.
To circumvent the problem of template folding and 3′-end self-priming, care has to be taken in defining the PCR conditions, mainly with respect to the length of the PCR product and the PCR primer positions on the target DNA. Pyrosequencing AB (Uppsala, Sweden), the manufacturer of the Pyrosequencing hardware, provides software for the design of sequencing primers, which includes the verification of the PCR product's sequence for “template looping onto itself.” Furthermore, the single-stranded fragment should not exceed 300 bases because the possibility of self-priming increases with template size. This is a limitation because, in many cases, repetitive sequences around the SNP to be analyzed require larger fragment sizes. The same may be necessary when analyzing cDNA where primers for PCR must span several exons, such as to amplify different splice forms with a single primer pair. Also, for quantitative methylation analysis of CpG sites by Pyrosequencing, the probability of forming 3′ loops after ssDNA preparation is high because the sequence is of low complexity after bisulfite treatment, consisting of essentially 3 bases only.
In such cases, the formation of secondary structures and occurrence of template self-priming have to be overcome by the experimental design. Pyrosequencing using double-stranded DNA (dsDNA) as a template has been previously described, but the incomplete removal of PCR reagents may induce background signals (2,4). Nordstrom et al. (2) therefore developed a protocol for dsDNA Pyrosequencing that included oligonucleotides blocked at their 3′ ends by a phosphate or an amino group and proposed the use of such modified oligonucleotides when sequencing with ssDNA templates forming 3′end loops.
Another possible solution is the addition of ssDNA binding protein (SSB) in the sequencing reaction (1,5,6,7,8,9,10). However, the binding of SSB to ssDNA varies depending on the DNA sequence and its size (1,11).
Here we present two possible ways to avoid the single-stranded template's self-annealing and priming by blocking its 3′ end. First, an oligonucleotide that is identical to the nonbiotinylated PCR primer but with a dideoxynucleotide at its 3′ end can be annealed to the template. This oligonucleotide competes with the ssDNA 3′ end for internal annealing sites and prevents the template's 3′ end self-annealing. We call this type of oligonucleotide a blOligo (blocking oligonucleotide), according to the designation coined by Nordstrom et al. (2), which cannot be extended during the sequencing reaction due to the presence of the dideoxy residue at its 3′ end. Second, the 3′ end OH of the template is enzymatically modified with terminal deoxynucleotidyl transferase (TdT; Amersham Biosciences) by incorporating a single dideoxynucleotide. Thus, the 3′ end of the modified template will not be elongated.