Thermodynamic factors influencing the application of DNA probes and DNA binding fluorescent dyes for detection of mutations and polymorphisms

This study has been conducted & submitted In Partial fulfillment of the requirement for the degree of Master of Science in Molecular Biology, Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia, 1428-1429H \ 2007-2008GThe Polymerase Chain Reaction (PCR) first described in the mid-1980s revolutionized molecular genetics by facilitating the rapid amplification of specific segments of DNA (cloning), enabling further analysis and functional use of such products. Two other fundamental technologies in molecular genetics, DNA sequencing and in vitro mutagenesis, are most often PCR based. The present investigation addresses the effect of using Real Time PCR and Real time assays based upon incorporation of DNA binding fluorescent dyes that have been used to monitor the thermodynamic stability of short double stranded DNA molecules. Two fluorescent dyes, SYBR Green and LC Green had been used for monitoring thermodynamic stability. During the investigation we studied many factors in relation to hybridization of DNA with particular reference to thermodynamic stability and affinity of DNA binding dyes. Real time assay formats based on hybridization depends on : the length of DNA fragments being analyzed, the position of alterations (polymorphisms or mutations) in sequence in relation to the overall strand (i.e. close to the middle or ends), the nature of the mismatch (G:C or A:T), the hydrogen bond number (GC/AT ratio) or size/sequence of probe used. For melt curve analysis the affinity of SYBR Green relative to LC Green is also a factor. This study indicates that genotypes with a specific base change (mutation) can be identified through this melt curve signature with LC GREEN I or SYRB GREEN I. The study identified the effects of amplicon length, sequence and GC content on the ability to identify the mutation. The effect of magnesium ion concentrations on the Real Time PCR assay was also studied. Specific products can be distinguished from non-specific products, eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than one hour. The assay described in the study provides several advantages for routine clinical testing for (Single Nucleotide Amplification) SNPs and detection of small genetic mutations. It offers high specificity and accuracy. The procedure is rapid, taking about an hour from PCR setup to data analysis. Because it requires no fluorescent labeling or sophisticated equipment, the system is inexpensive. Also, it is easy to design and perform in the system