Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

Abstract

Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing-based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to "tailor" DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated "dyad" pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost-effective DNA length measurement for STRs and resequencing of small DNA fragments.

Figure 1

Concept of DNA template tailoring. (A) Dyad pyrosequencing of GATA31E08 under standard conditions. As shown in panel A, it is not possible to interpret the sequence results due to the occurrence of unsynchronized polymerization of heterozygous individuals at this locus. In dyad pyrosequencing, each peak represents two bases (AG or AT) but the first peak in pyrogram, represents three bases (AGA) because third base (A) is also available for polymerization during dispension of two nucleotides “AG” in the repeat motif of this marker (AGAT). (B) Dyad pyrosequencing of GATA31E08 with ddNTP. Addition of ddCTP does not produce accurate genotypes in heterozygous samples. (C) Partial DNA template tailoring of GATA31E08 using PNA alone. Dyad pyrosequencing using PNA to signal the ends of the separate strands produces recognizable genotypes. Note that the PNA probe includes 4 base pairs complementary to the repeat region in addition to the portion that is complementary to the flanking region. Therefore, the genotype for this heterozygous sample is properly reported as [7, 12] instead of the apparent genotype 6, 11. (D) Full Template Tailoring of GATA31E08 using PNA with ddCTP. Use of PNA along with terminating nucleotides (in this case, ddCTP) reduces the signal-to-noise ratio and results in accurate determination of genotype by dyad pyrosequencing.

Figure 2

DNA template tailoring by using PNA and ddCTP for homozygous and heterozygous samples for the marker GATA172D05. (A) Partial template tailoring of homozygous samples using PNA alone (genotype 6, 6), In dyad pyrosequencing, each peak represents two bases (TA or GA). (B) Full template tailoring of homozygous samples using PNA and ddCTP (genotype 6, 6). (C) Sequencing of heterozygous sample without template tailoring (non-interpretable genotypes). (D) Partial template tailoring of heterozygous sample using PNA alone (genotype 6, 12). (E) Full template tailoring of heterozygous samples using PNA and ddCTP (genotype 6, 12).

Figure 3

Dyad pyrosequencing using PNA and ddCTP for homozygous and heterozygous samples for the marker GATA31E08. (A) Sample Ak09; samples were run blind. Pairs of nucleotides were pooled and dispensed for dyad pyrosequencing. Template tailoring of homozygous samples using PNA as well as ddCTP. The genotype for this sample is 7, 7 instead of apparent genotype 6, 6 as explained in Fig. 1. (B) Sample Ak08; samples were run blind. Pairs of nucleotides were pooled and dispensed for dyad pyrosequencing. Template tailoring of heterozygous samples using PNA as well as ddCTP. The genotype for this sample is [7, 12] instead of apparent genotype [6, 11],] as mentioned in Fig. 1.