应用甜菜碱增加富含三核苷酸重复的高G--C含量DNA片段测序辨识度的最佳浓度探讨

Investigation of optimum concentrations of betaine for improving the resolution of sequencing G-C rich DNA with trinucleotide repeats

目的通过在Sanger法测序体系中加入浓度梯度的甜菜碱，建立高辨识度的富含三核苷酸重复的高G-C含量DNA片段的测序体系。方法以G-C含量为72％无三核苷酸重复的Nogo-B基因cDNA的5’端序列（the 5’ terminal of Nago—BcDNA，Am-Nogo-B）及G-C含量为74％的富含CAG重复与CCG重复的Huntingtin基因cDNA的5’端序列（the 5’ terminal of huntingtin cDNA，Am-HTT）为例，在Sanger法测序体系中加入浓度梯度的甜菜碱，比较测序结果。结果Am—Nogo—B与Am-HTT基因测序体系中最适宜的甜菜碱浓度相差较大。甜菜碱浓度在0．8～1．2mol／L之间时，Am-Nogo-B测序结果质量最佳。甜菜碱浓度在1．6～2．4mol／L之间时，Am-HTT基因的测序质量最好。结果具有良好的可重复性。结论不同碱基组成类型的高G-C含量DNA片段，即使G-C含量接近，最适宜测序体系中甜菜碱浓度不同。探索出了适宜于增加富含三核苷酸串联重复的高Gc含量DNA片段测序辨识度的体系，可作为类似DNA片段测序的参考方法。

Objective To develop an optimal sequencing system which can improve the resolution of sequencing CrC rich DNA with abundant trinucleotide repeats by applying concentration gradients of betaine to the Sanger sequencing system. Methods Concentration gradients of betaine were introduced into the sequencing system by taking the 5＇ terminal of Nogo-B cDNA （Am-Nogo-B） （G-C% ： 72%, without trinucleotide repeats） and 5r terminal of Huntingtin eDNA （Am-HTT） （G-C% ： 74 %, with abundant CAG and CCG repeats） the results of sequencing were compared. Results The optimum concentration of betaine for sequencing Am-Nogo-B has differed from that for Am-HTT. Result of sequencing Am-Nogo-B has achieved the best quality when the concentration of betaine was at 0.8 - 1.2 mol/L, whereas the result of sequencing Am-HTT obtained the best quality when the concentration of betaine was at 1.6 - 2.4 mol/L. The results were reproducible. Conclusion G-C rich DNA with similar CJ-C% required different concentrations of hetaine in the sequencing system due to base pair compositions. The sequencing system developed for improving the resolution of sequencing of Cr-C rich DNA with abundant trinucleotide repeats can be used as a reference for similar studies.