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\centerline{\bf Table 3: Experimental protocols }

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\centerline{\bf Table 3a: HFE  amplification protocol for 
small amplicon melting analysis }

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DNA was extracted using a QIAamp DNA Blood Kit (QIAGEN,  Inc., Valencia, 
CA), concentrated by ethanol precipitation and quantified by A260.  

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For high-resolution melting analysis, we used small amplicon melting
with primers as close to the SNP as dimer and misprime constraints
permit, as described in Liew et al. (2004) $^{(14)}$.
The PCR protocol followed here was modified slightly from the 
protocol described in (12). The amplicon was 40bp 
long. PCR was performed in a LightCycler with reagents commonly used in 
clinical laboratories.  Ten microliter reaction mixtures consisted of 25ng 
of genomic DNA, 3 mo MgCl2, 1x LightCycler FastStart DNA Master Hybridization 
Probes master mix, 1x LCGreen Plus, 0.5 $\mu$M forward (CCAGCTGTTCGTGTTCTATGAT ) 
and reverse (CACACGGCGACTCTCAT) primers and 0.01U/$\mu$l Escherichia coli 
(E. coli) uracil N-glycosylase (UNG, Roche).  The PCR was initiated with a 
10 min hold at 50\degrees C for contamination control by UNG and a 10 min 
hold at 95\degrees C for activation of the polymerase.  Rapid thermal 
cycling was performed between 85\degrees C and the annealing temperature 
at a programmed transition rate of 20\degrees C/s for 40 cycles.  Samples 
were then rapidly heated to 94\degrees C and cooled to 40\degrees C 
followed by melting curve analysis between 60\degrees C and 85\degrees C to 
confirm the presence of amplicon. 

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Prior to analysis on the HR1, samples 
were again rapidly heated to 94\degrees C and cooled to 40\degrees C to 
promote heteroduplex formation. 

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Following amplification, an additional melting was performed to denature 
the perfectly complementary post-extension duplexes after which the 
temperature was rapidly decreased to re-anneal strands independent of 
the presence or absence of a single mismatched base-pair. 


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\centerline{\bf Table 3b: HFE  amplification protocol for Temperature-Gradient 
Capillary Electrophoresis (TGCE) analysis}

\bigskip

The PCR protocol followed here was modified slightly from the protocol 
described in Bernard et al. (1998) $^{(15)}$. The amplicon was 242bp long. 
PCR was performed in a Perkin Elmer 9700 block cycler with similar 
reagents to those used for amplification in the LightCycler.  Ten 
microliter reaction mixtures consisted of 25ng of genomic DNA, 
3 mM MgCl2, 1x LightCycler FastStart DNA Master Hybridization Probes 
master mix, 0.4 $\mu$M forward (CACATGGTTAAGGCCTGTTG) and reverse 
(GATCCCACCCTTTCAGACTC) primers and 0.01U/$\mu$l Escherichia coli (E. coli) 
uracil N-glycosylase (UNG, Roche). All samples were then overlayed 
with mineral oil to prevent evaporation. The PCR was initiated with a 
10 min hold at 25\degrees C for contamination control by UNG and a 6 
min hold at 95\degrees C for activation of the polymerase. Thermal cycling 
consisted of a 30s hold at 94\degrees C, a 30s hold at 62\degrees C and 
a 1min hold at 72\degrees C for 40 cycles followed by a 7min hold at 
72\degrees C for final elongation.  

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Upon completion of these thermal 
cycles the samples were then heated to 95\degrees C for 5min followed 
by a slow cool over approximately 60min to 25\degrees C. 

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\centerline{\bf Table 3c: Analysis protocol for 
Temperature-Gradient Capillary Electrophoresis (TGCE) }


\bigskip

The protocol followed here is similar to that described in 
Margraf et al. (2004) $^{(16)}$. To prepare samples for TGCE analysis, 
PCR amplicons were transferred to 24 well TGCE trays and diluted 1:1 
with 1xFastStart Taq polymerase PCR buffer (Roche). These samples were 
then overlayed with mineral oil and the trays loaded into the TGCE 
instrument. TGCE was performed on a commercial instrument 
(Reveal (TM-melting temperature or trademark?)
mutation discovery system, reagents and Revelation software by 
SpectruMedix LLC, State College, PA) $^{(17?)}$.  DNA samples were injected 
electro-kinetically at 2 kV for 45 seconds, resulting in peak heights 
ranging from 5,000-40,000 intensity units with ethidium bromide 
staining. Optimal results were obtained when the temperature was 
ramped from 60-65\degrees C over 21 minutes and data was acquired over 
35 minutes.  Sequential camera images were converted to plots of 
image frame number (time) versus intensity units (DNA concentration).


\bye

spectrumedix TM and reference?
