Effect Of Base Mismatch On DNA Structure And Melting Temperature(Tm)

ObjectiveGene microchip is a very important innovation of the 20th century. It has been widely used in many fields, but the accuracy has becoming the major hurdles in the development of gene microchip.Because the sequence and the length of the probes are different, the thermal stability of the hybrids are not in the same .In order to facilitate the design of the probes, we must have some insight to the properties of base-match.Although oligonucleotide probe complementary to single nucleotide substitutions are commonly used in microarry-based screens for genetic variation, little is know about the hybridization properties of these probes in order to improve the specificity and sensitivity of oligonucleotide microarry-based mutational analysis of disease-related genes.In this paper, the influence of hybridization conditions was studied in order to better discern between fully complementary and mismatched strands. The type and location of the single-base mismatch, as well as the influence of the length of the strands were studied too.Materials and Methods1. DNA oligonucleotidesThe DNA oligonucleotides were obtained from TAKARA Corporation, all of them were dissolved in Tris-HCl buffer(0.1M NaCl, PH 7.4), and stored at -20℃. 2 . Nucleic acids hybridization and Melting temperature (Tm)measuringThe concentrations of the probes and targets were 1μM. Melting temperature studies were performed by measuring the change in absorbance at 260nm using a Cary 100 Bio UV/Vis spectrophotometer (Varian Inc).3. PCR reactionWe used primers containing mismatches in PCR reaction to enhance discrimination of single-base mismatch .Results and Discussion1. The type of mismatch significantly affect the stability of the chimers, taking into account that the stability for base pairs obtained from statistical results follows the sequence: GT = GA > AA = TT > TC≥AC≥CC.2. The position of mismatch affect the stability of the chimers too, When the mismatch located at the terminal situation, the strands are more stable than mismatch located at the center of the strand.3. Duplexes containing a single base deletions or insertions are less stable than perfect-match probes, although subject to sequence context effects, duplexes containing a single base bugle are predicted to be more stable than those containing single nucleotide mismatch.ConclusionsBase-mismatch can lead to gene mutation, which is the fundamental reason of some disease in human being. Furthermore, little effort has been made to systematically access to hybridization properties of probes complementary to these base-mismatch. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray, and would facilitate the design of good probes .