Unlike the conventional PCR (40 cycles), touch-down and TU gradient PCRs have similar numbers of cycles, i.e., 50. In spite of using the same quantity of template DNA for all of the amplification reactions, the products generated by the touch-down gradient PCR were more intensely visualized by ethidium bromide staining compared with other protocols. For DOI1, all of the three amplification protocols produced more than one product and therefore, could be accounted for by the primer sequence. In this comparative analysis, conventional and touch-down PCRs produced specific products for seven and five out of 11 genes, respectively, while the touch-down gradient PCR produced specific amplification products for the 11 genes analyzed. 1A) was compared with conventional and touch-down PCRs ( Fig. The TU gradient amplification protocol ( Fig. The amplification products were separated on 2.5% agarose gels and compared with O'GeneRuler 100 bp DNA Ladder (Fermentas, York, UK). The 10-cycle loop was repeated five times (50 cycles). However, in TU gradient PCR, a 10-cycle loop, consisting of 95☌ for 30 s, 48☌ for 30 s (+0.5☌ every cycle), and 72☌ for 1 min, was carried out. The cycling conditions used for each of the protocols followed are: conventional PCR (40 cycles 95☌ for 30 s, 48☌ for 30 s, and 72☌ for 1 min) and touch-down PCR, followed by 40 cycles of 95☌ for 30 s, 48☌ for 30 s, and 72☌ for 1 min. HotStarTaq DNA polymerase required an initial activation step of 95☌ for 15 min before the start of the cycling. The primer sequences used in the current study were designed using MethPrimer and are as follows: HK2 5′-TTTTGGATTATTTGAGATTTTTGAGAT-3′ and 5′-ATTAGGGGATTGGTTTTTGGTT-3′ (292 bp) MTSS1 5′-GGGGTTTAAGGTATTGGTTGTAAGT-3′ and 5′-AAACTAACTTTCCCTCTCAATCTCC-3′ (208 bp) SVIL 5′-CGTTTGGTGGTTTAGTAGAGGGC-3′ and 5′-AAACTCGCGCGTCCCC-3′ (73 bp) TAZ 5′-GTTTATTTGGATTTTGGTTAGTAG-3′ and 5′-TACAACACCTCCCTATTATACAC-3′ (244 bp) LHX9 5′-AGGTTTTTTGTGTAGAGATGTGTT-3′ and 5′-AATACACCAAACTATCCTTCATACC-3′ (169 bp) BHLHE41 5′-GTTTGGAGTGAGAGTAAATTATTAG-3′ and 5′-TTAAAAAACCTTAAAAAAATCTATAC-3′ (180 bp) DIO1 5′-TGTATTAGTAGGTA AAGAAAAGAGTGT-3′ and 5′-CTCCCAAATAACTAAAATTACAAAC-3′ (206 bp) PTGDS 5′-TTTGTTAGGGGAAGGGTATTTTT-3′ and 5′-TATCCTAACACCCAAATATAAAAC-3′ (300 bp) MCJ 5′-AAGTATATAAAGTTTTTTGAGGGTT-3′ and 5′-CAAAACTCACCAATCTCTACTAATC-3′ (401 bp) EPHX1 5′-TATTGGGGGAAGGAGTTTGTAG-3′ and 5′-AAACAACCATATTACTCACA3CAAAAC-3′ (265 bp) NQO1 5′-GTTTTTGTAGGTTGTTTATTTTAAA-3′ and 5′-CCTCCACAAACACCAATACTC-3′ (258 bp).īisulfite-modified DNA (2 μl) was amplified using gene-specific primers and 1 U HotStarTaq DNA polymerase (Qiagen) in a 20-μL reaction volume, according to the manufacturer's instructions. To confirm the efficacy of this unique amplification technique promotor-associated CpG island of 11 genes-hexokinase-2 ( HK2), metastasis suppressor-1 ( MTSS1), supervillin ( SVIL), tafazzin ( TAZ), lim homeobox-9 ( LHX9), basic helix-loop-helix family member e41 ( BHLHE41), deiodinase, iodothyronine, type I ( DIO1), PGD 2 synthase ( PTGDS), DnaJ (Hsp40) homolog, subfamily C, member 15 ( MCJ), epoxide hydrolase 1, microsomal ( EPHX1), and NAD(P)H dehydrogenase, quinone 1 ( NQO1)-they were amplified in IN699, a short-term cell culture derived from a pediatric glioblastoma multiform. This technique has been designated as Touch-Up (TU) gradient PCR. Therefore, we report here a PCR amplification technique especially for bisulfite-modified DNA, which requires minimum optimization and produces specific products using a temperature range throughout the amplification process. In addition, some methods require double amplification, using nested primers, which increases the chances of nonspecific product generation. Irrespective of the amplification method used, all require prior optimization of the PCR to determine suitable annealing temperatures. Conventional PCR is rarely used, and specialized modifications of PCR, such as touch-down or nested PCRs, are used frequently. 4 However, the successful amplification of bisulfite-modified DNA is often challenging. These include methylation-specific PCR, 1 combined bisulfite restriction analysis, 2 methylation-sensitive single nucleotide primer extension, 3 and bisulfite sequencing. Some of these techniques require amplification of the bisulfite-modified DNA to study methylation at specific loci within the genome. Bisulfite-modified DNA is the primary requirement for almost all DNA methylation analysis methods available today.
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