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Fig. 2 (A-D) Comparative analysis of the hMLHl A655G SNP with PCR-RFLP and GALIOSTM. (A) Scheme of the PCR-RFLP strategy. A 191-bp PCR product amplified from allele A, but not from allele B, is cut into a 38- and a 153-bp fragment by Clal digestion. (B) Analysis of Clal restriction digestion by agarose gel electrophoresis. Lanes 1 and 9: 100-bp DNA ladder. Lane 2: negative control (n.c.). Lanes 3-8: PCR products from gDNAs of hMLH1 genotype A/A (3 and 4), A/G (5 and 6), or G/G (7 and 8) before (''-'': 3, 5, and 7) and after (''+'': 4, 6, and 8) Clal digestion. All samples show the expected restriction fragment pattern, i.e., 153 and 38 bp for A/A, 191, 153, and 38 bp for A/G, and 191 bp for G/G genotype. The 38-bp fragment is poorly visible on the gel. (C) Data generated by GALIOSTM and FCS analysis from reactions containing no DNA (n.c.) or gDNA samples of different hMLH1 A655G genotype (361-409). (D) Box plot illustrating the highly precise distribution of NFS values obtained from (n) gDNAs for each hMLH1 A655G genotype (A/A, A/G, and G/G). Median, S.D., and CI 99.99% values are denoted at the respective boxes. (E) Precision data (mean, S.D., and CI 99.99%) for intrasample, interwell, and intersample precision obtained from (n) determinations of each hMLH1 genotype. (View this art in color at www.dekker.com.)

Fig. 2 (A-D) Comparative analysis of the hMLHl A655G SNP with PCR-RFLP and GALIOSTM. (A) Scheme of the PCR-RFLP strategy. A 191-bp PCR product amplified from allele A, but not from allele B, is cut into a 38- and a 153-bp fragment by Clal digestion. (B) Analysis of Clal restriction digestion by agarose gel electrophoresis. Lanes 1 and 9: 100-bp DNA ladder. Lane 2: negative control (n.c.). Lanes 3-8: PCR products from gDNAs of hMLH1 genotype A/A (3 and 4), A/G (5 and 6), or G/G (7 and 8) before (''-'': 3, 5, and 7) and after (''+'': 4, 6, and 8) Clal digestion. All samples show the expected restriction fragment pattern, i.e., 153 and 38 bp for A/A, 191, 153, and 38 bp for A/G, and 191 bp for G/G genotype. The 38-bp fragment is poorly visible on the gel. (C) Data generated by GALIOSTM and FCS analysis from reactions containing no DNA (n.c.) or gDNA samples of different hMLH1 A655G genotype (361-409). (D) Box plot illustrating the highly precise distribution of NFS values obtained from (n) gDNAs for each hMLH1 A655G genotype (A/A, A/G, and G/G). Median, S.D., and CI 99.99% values are denoted at the respective boxes. (E) Precision data (mean, S.D., and CI 99.99%) for intrasample, interwell, and intersample precision obtained from (n) determinations of each hMLH1 genotype. (View this art in color at www.dekker.com.)

volume containing 5 ng gDNA. GALIOSTM reactions consisted of 1 x EVO^mp buffer (Evotec Technologies GmbH), 2.5 mM MgCl2, 0.2 mM of each dNTP, and 1 U Q-BioTaq (Q»BIOgene, Heidelberg, Germany). Amplification, diagnostic, and universal labeling primers were 300, 10, and 5 nM, respectively. Thermal cycling included an initial 2-min denaturation at 94°C, followed by 45 cycles of 94°C for 20 sec, 55°C for 40 sec, and 72°C for 45 sec. Samples were maintained at 72°C for 5 min before holding at 4°C prior to analysis. FCS measurements were performed for 3 x 3 sec in each reaction both at 543 and 633 nm. Mean values of the fractions of fluorescent PCR product (amplimer) from triplicate measurements were used for further genotype calculations. In all samples, amplimer values indicating allele-specific amplification signal were at least 8-fold higher than the corresponding background ''noise'' from no-DNA control reactions at the respective wavelength (SNR>8) (Fig. 2C). Bi-allelic SNP scores were made when the SNR was > 8 and the ratio of the higher to the lower allele signal was > 6 for homozygous and < 2 for heterozygous scores. Calculation of the NFS revealed distinct values for each hMLH1 genotype (A/A, A/G, and G/G), and the resulting bi-allelic SNP scores were 100% identical for both GALIOS™ and PCR-RFLP, demonstrating the accuracy of GALIOSTM genotyping. The high confidence of the GALIOSTM genotyping results was also confirmed by the narrow intragenotype distribution of NFS values (Fig. 2D).

Reproducibility and precision of GALIOSTM genotyping were assessed by repetitive hMLHl genotyping of subsets of gDNAs and evaluation of the distribution of the NFSs (Fig. 2E).

For intrasample precision, a single gDNA sample per hMLHl genotype was amplified and measured by FCS for

10 x 3 sec at both 543 and 633 nm. All combinations of individual amplimer values from both wavelengths were used to calculate 100 individual NFSs per genotype. For interwell precision, a single gDNA per hMLHl genotype was amplified in 10 individual GALIOSTM reactions. Each reaction was measured by FCS, each for 3 x 3 sec at both

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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