Solution-phase, homogeneous DNA melting analysis was performed in 100 nl volumes on a custom microchip/instrument and compared to the gold-standard 10 microliter HR-1™ melting instrument for the VKORC1 C1173T target, one of the genetic targets involved in determining warfarin dosing used in blood thinners.  Normal sample was distinguished from heterozygous and homozygous single-nucleotide polymorphisms (SNPs) simultaneously in an array melting platform using an automatic genotype calling melting analysis software.

Introduction. In 1997 DNA melting analysis was introduced¹, using a double-stranded DNA fluorescent dye to genotype or scan for SNPs following the polymerase chain reaction (PCR) for DNA amplification.  Analysis of the melting transition is often sufficient for genotyping. However, unlabeled probes combined with asymmetric PCR provide even greater specificity over a smaller region, which may be necessary for variant discrimination², as is the case with the specific target VKORC1 C1173T.  Probe detection specificity is commonly believed to be essential for clinical assays³.

Materials and Methods. Xurography⁴ was used to create a PDMS soft lithography mold, with analysis wells being 1.125 mm in diameter and 0.1 mm in height, creating a 100 nl volume.  PDMS was then molded and cured in an oven.  PDMS ports were cored and then bonded to glass slide substrates using corona discharge, Figure 1.  A Peltier heater and J-type thermocouple were used for temperature control.  Detection used a CCD camera (iXon, Andor Technology, South Windsor, CT) and LEDs with filters designed for the double-stranded dye LCGreen® Plus (Idaho Technology, SLC, UT), Figure 2.  All hardware was controlled using LabView 8.0 (NI, Austin, TX).  10X asymmetric PCR with unlabeled oligonucleotide probes was performed on the DNA sample using a LightCycler® (Roche Applied Science, Indianapolis, IN) and then transferred to an HR-1™ instrument (Idaho Technology) for a high-resolution reference melting curve.  The amplified sample was pipetted into the microchip for nanoliter melting analysis.  All three genotypes were melted simultaneously and each genotype had three wells for repeat analysis.

Results & Conclusion. Figure 3 shows negative derivative melt plots of normal, heterozygous, and homozygous SNPs within a 190-bp fragment of VKORC1 C1173T.  The 100 nl melts are in good agreement with the 10 microliter HR-1 melt curves and each of the genotypes were clearly distinguished in their first melt transition, the unlabeled probe melt region, using an automatic calling software (Idaho Technology) to distinguish the genotypes.  Previous research has shown that SNPs can be detected down to volumes of 10 nL with symmetric PCR⁵.  This platform uses ten times more volume because in theory asymmetric PCR provides ten times less amplification.  This new platform provides accurate SNP genotyping capability using a hundredth of the volume currently used with gold-standard instrumentation.  This technology with further development will lead to a more accurate warfarin dosing within the clinical setting.

References

1. K. Ririe, R. Rasmussen, and C. Wittwer Anal Biochem. 1997, 245, 154-160.
2. L. Zhou, L. Wang, R. Palais, R. Pryor, and C.T. Wittwer Clin Chem. 2005, 51(10):1770-77.
3. L. Zhou, A.N. Myers, J.G. Vandersteen, L. Wang, and C.T. Wittwer Clin Chem. 2004, 50(8):1328-35.
4. D. Bartholomeusz, R. Boutte, and J. Andrade J. MEMS 2005, 14:1364-74.
5. S.O. Sundberg, C.T. Wittwer, J. Greer, R.J. Pryor, O. Elenitoba-Johnson, and B.K. Gale Biomed Microdevices 2007, 9:159-166.

Figure 1.  Photograph of the custom microchip showing three channels with three wells per channel.  Each well has a diameter of 1.125 mm and a depth of 0.1 mm.

Figure 2.  Photograph of the detection instrumentation.  (1) CCD camera; (2) LED ring; (3) heating platform; (4) thermocouple control; and (5) LED power supply.

Figure 3. Negative derivative plots of 10 microliter melts (top) and 100 nl melts (bottom). Normal DNA (blue); heterozygous mutation (black); and homozygous mutation (red).