(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.
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.
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).