The accurate determination of Tm value was the basis in molecular biology experiment and the Tm value was very important in studying of gene chip.In this paper,proposing a method determined Tm value of DNA fragment by RT-PCR,which had the advantages of small amount of solution,simple operation,and determining the Tm value of the small fragment DNA Comparing with the traditional UV method.In addition,we builded a solution system containing Zn2+and betaine to make DNA fragments with different GC%content hybridizing at the same temperature.We optimized hybridization conditions of gene chip and provided a method for high throughput research of gene chip.1.The experimental conditions of RT-PCR method were studied to determine the Tm value of DNA fragments with different GC%content and different length.The solution conditions:DNA solution?final concentration 10ng/?L?,SYBR Green?1?L,phosphate solution?final concentration 5mmol/L?,Tris solution?final concentration60mmol/L?,the total experimental system was 40?L.The program of RT-PCR was 30?30s,65 cycles,adding 1?per cycle.Tm values of different GC%content and different length were lambda DNA 87?,150300bp with different GC%content?TPMT,CY,APOE?72?,78?and 90?,?Oligo,A,B,C?43?,67?,>90?).The experimental results were verified by UV method.Under the same experimental system,the Tm values by UV were lambda DNA 89?,150300bp with different GC%content?TPMT,CY,APOE?70?,79?and>90?.The method of RT-PCR could determine the Tm values of DNA fragments with different GC%content and different length and the results of RT-PCR were basically consistent with the UV method.2.A low viscosity solution system is presented for DNA hybridizing with different GC%content at the same temperature.On the basis of determining DNA melting curve by RT-PCR,we studied the difference of melting curve of DNA fragments with different GC%content after adding Zn2+and betaine.After investigating the influence of concentration on DNA melting tempratue,we chose10-44 mol/L10-22 mol/L of Zn2+concentration and 1.5 mol/L2.5 mol/L of betaine concentration in hybridization solution system,and using six DNA probes on gene chip as sample with different GC%content and mutation.Gene chip hybridized with target sequence under selecting conditions and setting the hybridization temperature as 31?.As result,combing 10-33 mol/L Zn2+concentration and 2.5 mol/L betaine eliminated the mutation of nonspecific probe hybridization signal,and the specific signal clear and uniform.DNA fragments with different GC%content could hybridize at the same temperature conditions using the low viscosity solution system. |