Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences Marco Musso, Renata Bocciardi, Sara Parodi,

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Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences Marco Musso, Renata Bocciardi, Sara Parodi, Roberto Ravazzolo, Isabella Ceccherini The Journal of Molecular Diagnostics Volume 8, Issue 5, Pages 544-550 (November 2006) DOI: 10.2353/jmoldx.2006.060058 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Amplification of RET promoter region. A: Nucleotide sequence (392 bp) spanning from −179 to +213 with respect to the transcription start site of RET gene. B: Base composition plot of GC content of the sequence. The amount and frequency of each deoxyribonucleotide and the percentage of GC are also indicated. C: Agarose gel electrophoresis. Ten μl from each PCR product obtained under different conditions, namely single additives or their combinations at a final concentration of 1.3 mol/L betaine, 5% DMSO, and 50 mmol/L 7-deaza-dGTP, were loaded as indicated (lanes 1 to 8). PhiX174 HaeIII digested (New England Biolabs) is indicated as a molecular weight marker. D: Chromatogram of the DNA sequence obtained with the RET f primer. Nucleotide number 147, corresponding to −5 promoter SNPs (G/A), shows the presence of two different alleles in the IMR-32 cell line, as expected. The Journal of Molecular Diagnostics 2006 8, 544-550DOI: (10.2353/jmoldx.2006.060058) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification of LMX1B. A: Nucleotide sequence (737 bp) spanning from intron 6 to intron 8 of LMX1B gene. B: Base composition plot of GC content; the amount and frequency of each deoxyribonucleotide and the percentage of GC are also indicated. C: Agarose gel electrophoresis. Five μl of each PCR, performed by using single additives or their combination at a final concentration of 1.3 mol/L betaine, 5% DMSO, and 50 mmol/L 7-deaza-dGTP, were loaded as indicated (lanes 1 to 8). PhiX174 HaeIII digested is indicated as a molecular weight marker. D: Chromatogram of the DNA sequence obtained with the LMX1B f primer. The Journal of Molecular Diagnostics 2006 8, 544-550DOI: (10.2353/jmoldx.2006.060058) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification of LMX1B. A: Nucleotide sequence (737 bp) spanning from intron 6 to intron 8 of LMX1B gene. B: Base composition plot of GC content; the amount and frequency of each deoxyribonucleotide and the percentage of GC are also indicated. C: Agarose gel electrophoresis. Five μl of each PCR, performed by using single additives or their combination at a final concentration of 1.3 mol/L betaine, 5% DMSO, and 50 mmol/L 7-deaza-dGTP, were loaded as indicated (lanes 1 to 8). PhiX174 HaeIII digested is indicated as a molecular weight marker. D: Chromatogram of the DNA sequence obtained with the LMX1B f primer. The Journal of Molecular Diagnostics 2006 8, 544-550DOI: (10.2353/jmoldx.2006.060058) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Amplification of PHOX2B. A: Exon 3 nucleotide sequence (627 bp) of the PHOX2B gene. AvaII restriction sites are in bold characters, the 21-bp sequence deleted in the control polymorphic variant are underlined, whereas the 18-bp sequence duplicated in the CCHS patient is shown in italic. B: Plot of GC content; the amount and frequency of each deoxyribonucleotide and the percentage of GC are also indicated. C: Agarose gel electrophoresis. Five μl of each PCR product obtained using genomic DNA from a control individual (lanes 1 and 2), from a control individual with the polymorphic deletion (lanes 3 and 4) or from a CCHS patient with an expanded allele (lanes 5 and 6), without any additive (lanes 1, 3, and 5) or with betaine, DMSO, and 7-deaza-dGTP (lanes 2, 4, and 6). D: Polyacrylamide gel electrophoresis of AvaII-digested PCR products from a control individual (lane 2), a control individual carrying the polymorphic deleted allele (lane 3), or a CCHS patient carrying the expanded allele (lane 4). Molecular weight markers are PhiX174 HaeIII digested and a 100-bp ladder (lanes 1 and 5, respectively). Arrows indicate the AvaII fragments containing the 18-bp expansion (288 bp) or the 21-bp deletion (249 bp) and the normal allele (270 bp). E: Chromatograms of DNA region involved in deletion or expansion whose origin is indicated by arrows. The Journal of Molecular Diagnostics 2006 8, 544-550DOI: (10.2353/jmoldx.2006.060058) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions