In this report we present spectroscopic evidence on
In this report, we present spectroscopic evidence on the effect of different solvents having proton acceptor and proton donor ability and pH of the medium on the abstraction of amino proton of PI (Scheme 1) for controlling the nonradiative rates. Despite the general utility of PI as a DNA intercalating probe, its interaction with biomimicking self-assembled systems (e.g. micelles, DNA, etc.) is only discretely studied so far , . A complete study on the behavior of the dye in different solvents as well as in biomimicking systems and DNA is firmly demanding. In this article, we have initiated a series of photophysical studies on the interaction of PI with micelles and fish sperm DNA by steady state and time resolved spectroscopy. The structural integrity of DNA in presence of the probe has also been confirmed by circular dichroism (CD) spectroscopy. All these studies are important to point out that fluorescent dye PI can be used for DNA-recognition in biologically relevant macromolecular environment.
Materials and methods
Results and discussion
DNA typing technologies using short tandem repeats allow comparisons to be made between body fluid stains and individuals. In cases where the presence of a suspect is expected, the type of body fluid present can make the distinction between innocent and criminal contact. In previous work, we have shown that analysis of DNA methylation patterns can identify the tissue source of a DNA sample , . DNA methylation is a natural process in the mammalian genome that involves the addition of a methyl group to the 5′ carbon of cytosines in a dinucleotide CpG pair. DNA methylation is involved in gene expression by regulating transcription factors that lead to gene activation or gene silencing . Several reports demonstrate that specific regions of the genome have different DNA methylation patterns depending on the cell type studied , , . Those regions are called tissue-specific differentially methylated regions and can be used as a powerful tool for body fluid identification , , . Common methodologies for DNA methylation analysis include DNA Trimidox by methylation-sensitive endonucleases followed by polymerase chain reaction (PCR) amplification and bisulfite conversion of genomic DNA that causes the unmethylated cytosines to be chemically converted to uracil while the methylated cytosines are protected. After bisulfite modification, the DNA is amplified by PCR using specific primers and the amplicons can be analyzed by pyrosequencing, which provides quantitative methylation values for each CpG site present in the target sequence . High-resolution melt (HRM) analysis can detect DNA sequence variants based on their different melting temperatures , , . PCR products can be differentiated even if they differ from each other at only a single nucleotide , making this an optimal method to analyze single nucleotide polymorphisms , , . HRM involves the amplification of a DNA template by real-time PCR in the presence of a double-stranded DNA (dsDNA) intercalating dye such as EvaGreen. The fluorescence is maximized at the end of amplification when the largest quantity of dsDNA is present. Once the PCR is complete, the melting step begins, whereby PCR products are heated in increments of 0.1 °C and a graph illustrating the change in fluorescence with respect to changes in temperature (–d/d) is obtained . HRM is an in-tube method, meaning that analysis occurs in the same tube as amplification, thereby saving time and avoiding sample transfer steps. The procedure is a quick and nondestructive method to characterize PCR products . HRM can also be used to explore the melting differences between unmethylated and methylated DNA after bisulfite conversion. During bisulfite conversion, the unmethylated cytosines, but not the methylated ones, are converted into uracils and subsequently thymine during PCR. Thus, amplicons resulting from the PCR of unmethylated DNA have a lower GC content and concomitant lower melting temperature when compared with amplicons resulting from the PCR of methylated DNA. If the bisulfite conversion fails, the GC content of the amplicon is similar to methylated DNA and an overestimation of methylation occurs , . Even though the available commercial kits for bisulfite conversion modify 99% of the DNA, appropriate controls should be in place to confirm that there is no amplification of unmodified DNA. One way of guaranteeing this step is to design primers that anneal only to bisulfite-modified DNA.