Surface Melting Curve Analysis with R

Nucleic acid Melting Curve Analysis is a powerful method to investigate the interaction of double stranded nucleic acids. Many researchers rely on closed source software which is not ubiquitously available, and gives only little control over the computation and data presentation. R in contrast, is open source, highly adaptable and provides numerous utilities for data import, sophisticated statistical analysis and presentation in publication quality. This article covers methods, implemented in the MBmca package, for DNA Melting Curve Analysis on microbead surfaces. Particularly, the use of the second derivative melting peaks is suggested as an additional parameter to characterize the melting behavior of DNA duplexes. Examples of microbead surface Melting Curve Analysis on fragments of human genes are presented. Introduction Melting Curve Analysis Nucleic acid Melting Curve Analysis (MCA) is a central step in nucleic acid1 interaction studies, identification of specific DNA sequences after quantitative real-time PCR, genotyping or detection of Single Nucleotide Polymorphisms2 (SNP) both in solution and on surfaces (Ririe et al., 1997; Gundry et al., 2003; Sekar et al., 2005; Zhou et al., 2005; Rödiger et al., 2012b). A review of the literature revealed that there is an ongoing demand for new bioanalytical devices to perform MCAs. This includes lab-on-chip systems or the recently published VideoScan platform (Rödiger et al., 2012b, see following section). Some of these systems offer software solutions for MCA but often custom made software is required. Although bioanalytical devices break new ground to meet criteria like high multiplex levels or new detection-probe-systems the fundamental concept of MCA remains unchanged. The MCA is a real-time monitoring of a heat-induced double stranded nucleic acid dissociation which can be monitored by the change of the referenced mean/median fluorescence intensity (MFI)3 at a defined temperature (Figure 1). 65 70 75 80 85 − 1. 0 − 0. 5 0. 0 T [°C] − re fM F I( T ) | r ef M F I'( T ) | r ef M F I'' (T )