Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene

Abstract

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min.

Figure 1

Representative electropherograms of p53 exon 8 wild-type (a) and wild-type + mutant samples for an easily identified mutation (b) and a more challenging mutation (c), with the detection of the mutations highlighted by . Mutations were detected by tandem SSCP/HA on a microfluidic device using the following conditions: ambient temperature, 2-color laser induced fluorescence (LIF) detection ( for the forward strand, for the reverse strand), 0.1% PHEA dynamically coated channel, 350 V/cm applied electric field strength. Baselines adjusted to value of zero to remove background noise.

Figure 2

Electropherograms of p53 exon 8 demonstrating temporal stability of SSCP and HA conformers that allowed mutation detection for at least 16 days after initial heat denaturing and snap cooling when stored at 0–4 °C. Detection of the mutations by tandem SSCP/HA is highlighted by . Mutations were detected on a microfluidic device using the following conditions: ambient temperature, 2-color LIF detection ( for the forward strand, for the reverse strand), 0.1% PHEA dynamically coated channel, 350–450 V/cm applied electric field strength. Baselines adjusted to value of zero to remove background noise.

Figure 3

Electropherograms of p53 exons 5–9 wild-type and wild-type + mutant samples demonstrating ability to detect mutations at mutant DNA concentrations as low as 15–18% mutant DNA with detection of the mutations by tandem SSCP/HAhighlighted by . Mutations were detected on a microfluidic device using the following conditions: ambient temperature, 2-color LIF detection ( for the forward strand, for the reverse strand), 0.1% PHEA dynamically coated channel, 350–450 V/cm applied electric field strength. Baselines adjusted to value of zero to remove background noise.