\magnification = \magstep1
\nopagenumbers


\centerline{\bf Figure 1. Distinguishable and Indistinguishable Genotypes }


\bigskip
When the wild-type and mutant homozygous genotypes are
symmetric with respect to nearest-neighbor thermodynamics,
their normalized melting curves are indistinguishable. 
This figure shows three replicate normalized melting curves
of each of the three genotypes of the H63D (Hemochromatosis)
mutation, which obeys this symmetry. Six curves representing
the wild-type and mutant homozygous genotypes appear as
one curve, while the normalized melting curves of the heterozygote 
appear as another easily distinguishable curve.

\vfill\eject

\centerline{\bf Figure 2. Theoretical duplex melting curves, superpositions and differences }

\bigskip

After amplification of heterozygous DNA (or a mixture of any
two genotypes of a biallelic SNP) two types of forward strands
reassociate independently with two types of reverse strand,
forming two species of homoduplex and two species of heteroduplex.
A fluorescent dye gives a signal proportional to the quantity of 
DNA which is in its double-stranded state, which depends on
the temperature and the thermodynamic stability of each
duplex type.  The overall fluorescence vs. temperature `melting 
curve' is a superposition of the melting curves of the individual 
duplexes in proportion to their relative concentrations.

\bigskip
This figure gives the melting curves for the four duplex species
of heterozygous or mixed genotype H63D amplicons as predicted
by nearest-neighbor theory model with parameters described
in (SL match and CC,GG). The wild-type and mutant homozygous
curves are identical. It also shows the theoretical heterozygote
melting curve obtained as the equally weighted average of
the four duplex curves. In the nearest-neighbor symmetric case, 
the difference between any genotype mixture melting curves is 
proportional to a standard difference curve which is the difference 
between the average homoduplex curve and the average heteroduplex curve,.
by a factor equal to the absolute difference in heteroduplex content.
Thus,  the difference between the 
heterozygote melting curve and the overlapping homoduplex curves,
which is also shown, is equal to the standard difference curve 
scaled by ${1 \over 2}$, the heteroduplex fraction of an amplified 
heterozygote.


\vfill \eject

\centerline{\bf Figure 3. Theoretical Dependence Of Heteroduplex Content On Mixture Fraction }

\bigskip

This figure shows the magnitude of the heteroduplex content differences
amomg mixtures obtained by adding wild-type DNA to DNA of each of the 
three genotypes of a bi-allelic SNP (W=wild-type, M=mutant homozygous, 
H=heterozygous) in the relative proportions $x$ of added wild-type 
(the `mixture fraction') to $1-x$ of the varied genotype. We assume
that strands of different types associate independently after either
PCR amplification is completed or a subsequent dissociation by heating
and reassociation by cooling have been performed. We seek the 
mixture fraction providing the greatest separation of heteroduplex 
content between the resulting mixtures, represented by the highest
point on the lowest among the three graphs. This point which is
marked at $x={1 \over 7}$, gives the optimal mixture fraction.


\vfill \eject

\centerline{\bf Figure 4. Experimental Melting Curve Separation Dependence On Mixture Fraction }

\bigskip

This figure shows the mean and standard deviation (error bars) of
the experimentally measured maximum melting curve separation
between replicates of mixtures of wild-type DNA with either 
mutant heterozygous or heterozygous samples, and the mean of the 
wild-type replicates, as a function of the mixture fraction. The 
values are normalized by a factor which makes the difference for an 
unmixed heterozygous sample equal to ${1 \over 2}$ to agree with the 
theoretical prediction in terms of heteroduplex content (superimposed).

\vfill\eject

\centerline{\bf Figure 5a. Genotype Separation At Optimal Mixture Fraction 4/28 }

\bigskip

This figure shows the normalized melting
curves for three replicates of optimal mixtures of
wild-type DNA and each genotype of sample. As predicted, the
mutant homozygous curves lie equidistant from the wild-type
curves and the barely altered heterozygous curves.


\vfill\eject

\centerline{\bf Figure 5b.  Genotype Separation At Non-Optimal Mixture Fraction 9/28 }

\bigskip

\centerline{\bf Figure 5c.  Genotype Separation At Non-Optimal Mixture Fraction 14/28 }

\bigskip

\centerline{\bf Figure 5d.  Genotype Separation At Non-Optimal Mixture Fraction 19/28 }

\bigskip


These figures show the normalized 
melting curves for three replicates of other-than-optimal mixtures 
of wild-type DNA and each genotype of sample. 
In Figure 5b, with mixture fraction is ${9 \over 28}$ near $x={1 \over 3}$, 
the mutant homozygous curves and the heterozygous curves have
become indistinguishable, as predicted.
In Figure 5c, with mixture fraction is ${14 \over 28}$,
i.e., wild-type and sample DNA mixed in equal 
proportions), the mutant homozygous curves and the heterozygous curves 
are also difficult to distinguish. 
In Figure 5d, with mixture fraction is ${19 \over 28}$ 
near the upper local maximum $x={2 \over 3}$, the mutant homozygous 
curves and the 
heterozygous curves have reversed position and are
distinguishable, but not nearly as well as at the optimal mixture fraction.

\vfill\eject

\centerline{\bf Figure 6a. Experimental Difference Curves Of Pure Genotypes }

\bigskip

\centerline{\bf Figure 6b. Experimental Difference Curve Of Optimally Mixed Genotypes }

\bigskip

These figures show the difference of normalized melting
curves of unmixed and optimally mixed samples of
the three genotypes and the mean of the wild-type
normalized melting curve replicates. The shape 
of the curves reflects the predicted standard
difference curve shape, and the amplitude agrees
with the predicted dependence on heteroduplex
content difference.

\vfill\eject

\centerline{\bf Figure 7a. Raw TGCE Duplex Arrival Frequency Data }

\bigskip

\centerline{\bf Figure 7b.  Normalized TGCE Duplex Arrival Frequency Data }

\bigskip

These figures show the raw and normalized measurements of 
duplex arrival frequency (counts per frame) for three 
replicates of each genotype and wild-type DNA with mixture 
fraction $x={9 \over 28}$. After normalization by shifting
and scaling the largest peaks to a common location and
height, the common heteroduplex content of the mutant
homozygous and heterozygous sample mixtures for this
mixture fraction are visible from the common first and
second heteroduplex peak heights.

\vfill\eject

\centerline{\bf Figure 8.  Heteroduplex Content Dependence On Mixture Fraction From TGCE }

\bigskip

This figure shows the mean and standard deviation (error bars) of
the experimentally derived heteroduplex content of
replicates of mixtures of wild-type DNA with either mutant heterozygous
or heterozygous samples, as a function of the mixture fraction. The 
values are adjusted by a factor which makes the value for 
control heterozygous samples equal to ${1 \over 2}$ to agree with 
the theoretical prediction of heteroduplex content 
(superimposed).


\end
