DNA samples.  Most of the samples used in this study were blood specimens  submitted to ARUP (Salt Lake City, UT) for routine clinical genotyping of prothrombin,  factor V, MTHFR, or HFE mutations.  DNA was usually extracted with the MagNa Pure  instrument (Roche Indianapolis, IN) according to the manufacturer’s instructions.   Additional samples genotyped at the β-globin locus for HbS were provided as dried  bloodspots by Pediatrix Screening Inc. (Pittsburgh, PA) and extracted as previously  described (13).  All samples were genotyped at ARUP or Pediatrix Screening by melting  curve analysis on the LightCycler  (Roche) using adjacent hybridization probe  (HybProbe) technology, using either commercial kits (Roche), or in-house methods (2,  11, 12).  At least three different individuals of each genotype for prothrombin  20210G>A, MTHFR 1298A>C, HFE 187C>G and β-globin 17A>T SNPs were selected.   One hundred and four samples (35 wild type, 35 heterozygote, 34 homozygous mutant)  previously genotyped for factor V (Leiden) 1691G>A were obtained.  Three rare DNA  samples with mutations near factor V Leiden (1691G>A) were also studied.  The first  sample was heterozygous 1690delC, the second was heterozygous 1690C>T and the third  was compound heterozygous 1696A>G and 1691G>A.  These samples were originally  identified by aberrant melting profiles during adjacent hybridization probe genotyping  and confirmed by sequencing.  All samples were de-identified according to a global  ARUP protocol under IRB #7275.    DNA samples obtained with the MagNa Pure or from dried blood spots were not  routinely quantified, but contained approximately 10-50 ng/μl.  However, for HFE 
187C>G genotyping, DNA was extracted using a QIAamp DNA Blood Kit (QIAGEN,  Inc., Valencia, CA), concentrated by ethanol precipitation and quantified by A260.  

The effects of ionic strength and product concentration on amplicon Tm have  been discussed previously (7).  Because homozygous SNP genotypes are distinguished  only by Tm, these issues are a concern for accurate genotyping.  However, the effect of  amplicon concentration on Tm is small and usually dwarfed by the temperature  (in)accuracy of most melting instruments.  Furthermore, by amplifying well into the  plateau phase, any differences in initial product concentration are usually equalized by  PCR.  Because Tm is strongly dependent on the ionic strength, it is important that all  DNA samples are extracted in the same way and end up in the same buffers.  We did not  find it necessary to quantify the DNA samples before PCR unless spiking studies were  performed and never attempted to quantify amplicon after PCR but before melting.  

13.  Heath EM, O'Brien DP, Banas R, Naylor EW, Dobrowolski S. Optimization of an  automated DNA purification protocol for neonatal screening. Arch Pathol Lab  Med 1999;123:1154-60.  

7.  Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT. Amplicon  melting analysis with labeled primers: a closed-tube method for differentiating  homozygotes and heterozygotes. Clin Chem 2003;49:396-406.