DAPK1

AcronymDefinition
DAPK1Death-Associated Protein Kinase 1
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In the pediatric group, 7 CpG island loci (CDH1, DAPK1, CRABP1, GR1N2B, T1MP3, CALCA, and TW1ST1) showed significantly higher methylation levels in results from H pylori-positive samples than those from H pylori-negative samples (all values, P < .
For a preliminary test of the reliability and performance of the MS-LAMP assay without the variation introduced by bisulfite treatment, we performed experiments to assess specificity, the detection limit, and selectivity with plasmid targets containing the DAPK1 promoter sequence in either a methylated state or a unmethylated state after bisulfite treatment.
We used 1 or 2 [micro]L (approximately 25-50 ng) of DNA as the target for investigating the hypermethylation status of the CDKN2A, DAPK1, and GATA5 promoters.
MS-LAMP primers (F3, B3, FIP, BIP, LF, LB), which specifically amplify only the methylated sequences of the CDKN2A, DAPK1, and GATA5 promoters after bisulfite treatment, were designed with the aid of PrimerExplorer V4 (Eiken).
For the multiplex LAMP reactions assessed by fluorescence, the LF primers of the DAPK1 and CDKN2A assays were substituted by oligonucleotides that had the same DNA sequence but were labeled at the 5' end with BODIPY FL (dipyrromethene boron difluoride FL) and 6-carboxytetramethylrhodamine (TAMRA) dyes, respectively.
The thermocycling protocols for the PCR reactions were as follows: CDKN2A, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 65 [degrees]C for 30 s, and 72 [degrees]C for 30 s; DAPK1, initial denaturation at 94 [degrees]C for 2 min and 45 cycles of 94 [degrees]C for 30 s, 68 [degrees]C for 30 s, and 72 [degrees]C for 30 s; GATA5, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 66 [degrees]C for 30 s, and 72 [degrees]C for 30 s.