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Locking of 3′ ends of single-stranded DNA templates for improved Pyrosequencing™ performance
 
Michael Utting1, Jochen Hampe2, Matthias Platzer1, Klaus Huse1
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(Figure 1) illustrates the self-priming problem and the two possible solutions. Ideally, no secondary structures interfere with the sequencing reaction ((Figure 1)B). Deoxynucleotides are attached only to the sequencing primer, and the correct sequence readout is obtained. If the template's 3′ end interacts with an internal complementary sequence ((Figure 1)A), nucleotides are incorporated at its 3′ OH, which falsify the sequence readout because the resulting signals overlay with the sequencing primer elongation. With the use of blOligo, the formation of secondary structures is prevented, giving rise to a correct sequence readout ((Figure 1)C), as is the case with TdT treatment, where the 3′ end of the template is locked ((Figure 1)D).

Materials and Methods

Allele-Specific Expression of CARD15

Total RNA from leucocytes was isolated using the RNeasy® kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the rtPCR oligo dt™ kit (Qiagen), according to the manufacturer's recommendations. PCR was performed with primers whose annealing sites were localized within exons 2 and 4 of caspase recruitment domain family, member 15 (CARD15), respectively. As a control experiment, the PCR products of CARD15 exon 2-exon 4 fragments were cloned into the PCR2.1TOPO™ vector (Invitrogen, Karlsruhe, Germany), and the recombinant vector was used as a PCR template instead of cDNA. The calculation of allele amounts occurs in reference to the common G ((Figure 2)A, labeled with an asterisk).

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.

Allele Frequency of A2M SNP rs226379

For the determination of allele frequency of α-2-macroglobulin (A2M) SNP rs226379, PCR with pooled genomic DNA (from 80–100 Caucasian individuals; Roche, Mannheim, Germany) was performed according to the manufacturer's instructions. For the calculation of allele frequencies, the PSQ™ 96MA 2.1 software (Pyrosequencing AB) was used.

PCR Conditions

Amplification was done in 25 µL containing MasterAmp™ 2× PCRPremix-Buffer D (Biozym Scientific GmbH, Oldendorf, Germany), 1 U of Taq DNA polymerase (Amersham Biosciences, Freiburg, Germany), 4 pmol of each primer, and with the following conditions: 1 cycle of 96°C for 5 min, followed by 45 cycles of 96°C for 30 s, 59°C for 35 s, and 72°C for 35 s, with a final cycle of 72°C for 5 min. PCR primer pairs used are listed in (Table 1).

Table 1. Oligonucleotide Sequences


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

aForward PCR primers.

Pyrosequencing

Biotin-labeled PCR products were immobilized on 10 µL streptavidin-coated Dynabeads® M280 (Dynal, Oslo, Norway) by mixing with 20 µL of PCR product and 30 µL 2× BW buffer II (Pyrosequencing AB). The samples were incubated by shaking at 43°C for 30 min, and afterwards they were transferred into 50 µL 0.3 M NaOH using the Multi Magnet PSQ 96 Sample Prep Tool (Pyrosequencing AB). The samples were washed in 100 µL washing buffer (Pyrosequencing AB) for 1 min and transferred into 40 µL annealing buffer, containing 4 pmol of sequencing primer, and kept at 80°C for 5 min. After equilibration to room temperature, the sequencing reaction was performed with the PSQ 96 SNP Reagent Kit, according to the manufacturer's directions, on a PSQ 96MA machine (both from Pyrosequencing AB).

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