Study On Determination Of DNA Melting Temperature And Improvement Of Chip Hybridization Difference

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/μL）,SYBR GreenⅠ1μL,phosphate solution（final concentration 5mmol/L）,Tris solution（final concentration60mmol/L）,the total experimental system was 40μL.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.