Locking of 3′ ends of single-stranded DNA templates for improved Pyrosequencing™ performance

Figures & data

Figure 1.  The self-priming problem in Pyrosequencing and two methods of circumventing the problem.

(A) Elongation of single-stranded DNA (ssDNA) template 3′ end due to self-priming. The formation of a secondary structure of the template causes the elongation of the sequencing primer (green) as well as of the template's 3′ end (red arrowheads). An incorrect sequence readout will result. (B) Ideal performance of Pyrosequencing without 3′ end self-priming, where only the sequencing primer is elongated. (C) Sequencing reaction in the presence of a blOligo (blocking oligonucleotide) designed to avoid self-priming of the template's 3′ end. The blOligo anneals to the 3′ end (blue arrowhead) of the template and, because it has a ddNMP at its own 3′ end (green dot), cannot be elongated. (D) Sequencing reaction of a template with ddNMP-modified 3′ end. Prior to sequencing, the template's 3′ end was modified by a deoxynucleotidyl transferase (TdT)-catalyzed addition of a dideoxynucleotide that will not be elongated even if self-annealing takes place. (B–D) A correct sequence readout is obtained.

Figure 2.  Characterization of a coding single nucleotide polymorphism (SNP) on CARD15 transcripts by Pyrosequencing.

SNP rs2067085 was analyzed on cDNA from leucocytes of three genotypically different individuals. (A) Theoretical histograms for the two homozygotes and the heterozygote with the chosen pipeting scheme. The histogram for the heterozygote was calculated for an equal expression of both alleles. Bases informative for the SNP alleles are shown in blue while bases in common are shown in brown. (B) Experimental Pyrograms obtained by using the standard protocol and cDNA from leucocytes. (C) Experimental Pyrograms obtained by using the standard protocol and cloned PCR products. (D) Results from blank control experiments in which Pyrosequencing was performed with single-stranded template without the addition of sequencing primer. (E) Blank control as in row D with the addition of blOligo (blocking oligonucleotide). (F) Blank control as in row D with the template ddCMP modified by deoxynucleotidyl transferase (TdT) treatment. (G) Pyrograms obtained by using blOligo and sequencing primer. (H) Pyrograms obtained by using ddCMP-modified template and sequencing primer. Red arrows indicate allele-specific bases used, and the asterisks (*) indicate the common G that was used as a reference for the calculation of allele amounts. CARD15, caspase recruitment domain family, member 15.

Table 1.  Oligonucleotide Sequences

Purpose	Target	Sequence
PCR	CARD15 ^a	5′-biotin-CGTCTCTGCTCCATCATAGG-3′
	CARD15	5′-TGGGAGCGGGGTTTCCTCAG-3′
	A2M ^a	5′-biotin-GGACTCTAGGTTCATGCTTCAC-3′
	A2M	5′-TTAGCCCGCCAGGAATTAAC-3′
Pyrosequencing	SNP rs2067085	5′-GTTGCCGATCTTCACACCGT-3′
	CARD15
	SNP rs226379	5′-TTCCACCCACACAAG-3′
	A2M
Blocking Self-Priming	CARD15	5′-TGGGAGCGGGGTTTCCTCAGddC-3′
	A2M	5′-TTAGCCCGCCAGGAATTAACddC-3′

CARD15, caspase recruitment domain family, member 15; A2M, α-2-macroglobulin.

a Forward PCR primers.

Table 2.  Analysis of Allele-Specific Expression of CARD15 in Leucocytes of Four Individuals

	Template	Allele Frequency G	Allele Frequency C	G:C	ΣG + C
Individual 1	standard	0.62	0.48	1.29	1.10
	blOligo	0.38	0.72	0.52	1.10
	ddNMP	0.39	0.65	0.60	1.04
Individual 2	standard	0.72	0.49	1.50	1.21
	blOligo	0.57	0.58	0.98	1.15
	ddNMP	0.46	0.57	0.81	1.03
Individual 3	standard	0.81	0.54	1.50	1.35
	blOligo	0.47	0.58	0.81	1.05
	ddNMP	0.48	0.52	0.92	1.00
Individual 4	standard	0.88	0.58	1.52	1.46
	blOligo	0.53	0.68	0.78	1.21
	ddNMP	0.52	0.54	0.96	1.06

Values were calculated through the analysis of single nucleotide polymorphism (SNP) rs2067085 on caspase recruitment domain family, member 15 (CARD15) cDNA by Pyrosequencing. For each individual, Pyrosequencing was performed using the standard procedure and after locking the 3′ ends of PCR products with the CARD15 blOligo (blocking oligonucleotide). Bold numbers indicate optimal Pyrosequencing performance after deoxynucleotidyl transferase (TdT) treatment.

Figure 3.  Determination of allele frequencies of the A2M gene single nucleotide polymorphism (SNP) rs226379 in pooled genomic DNA.

The sequence around SNP rs226379 was analyzed by the Pyrosequencing of a single sample containing pooled genomic DNA. (A) Theoretical histograms for the genotypes determined by SNP rs226379. The positions of the bases informative for the SNP are highlighted. (B) Pyrogram of a blank control experiment of single-stranded template without sequencing primer. (C) Pyrogram obtained using the standard protocol. (D) Pyrogram obtained using the α-2-macroglobulin (A2M) blOligo (blocking oligonucleotide). (E) Pyrogram using ddCMP-modified template. Red arrows indicate allele-specific base. The blue insets show the allele frequencies calculated from the peak heights.