中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2014年
2期
163-169
,共7页
甜菜碱%三核苷酸重复%高G-C含量%DNA测序
甜菜堿%三覈苷痠重複%高G-C含量%DNA測序
첨채감%삼핵감산중복%고G-C함량%DNA측서
Betaine%Trinucleotide repeats%G-C rich%Sequencing
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-B cDNA,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0.8～1.2 mol/L之间时,Am-Nogo-B测序结果质量最佳.甜菜碱浓度在1.6～2.4 mol/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G-C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.
目的 通過在Sanger法測序體繫中加入濃度梯度的甜菜堿,建立高辨識度的富含三覈苷痠重複的高G-C含量DNA片段的測序體繫.方法 以G-C含量為72％無三覈苷痠重複的Nogo-B基因cDNA的5'耑序列(the 5 ' terminal of Nago-B cDNA,Am-Nogo-B)及G-C含量為74％的富含CAG重複與CCG重複的Huntingtin基因cDNA的5'耑序列(the 5'terminal of huntingtin cDNA,Am-HTT)為例,在Sanger法測序體繫中加入濃度梯度的甜菜堿,比較測序結果.結果 Am-Nogo-B與Am-HTT基因測序體繫中最適宜的甜菜堿濃度相差較大.甜菜堿濃度在0.8～1.2 mol/L之間時,Am-Nogo-B測序結果質量最佳.甜菜堿濃度在1.6～2.4 mol/L之間時,Am-HTT基因的測序質量最好.結果具有良好的可重複性.結論 不同堿基組成類型的高G-C含量DNA片段,即使G-C含量接近,最適宜測序體繫中甜菜堿濃度不同.探索齣瞭適宜于增加富含三覈苷痠串聯重複的高G-C含量DNA片段測序辨識度的體繫,可作為類似DNA片段測序的參攷方法.
목적 통과재Sanger법측서체계중가입농도제도적첨채감,건립고변식도적부함삼핵감산중복적고G-C함량DNA편단적측서체계.방법 이G-C함량위72％무삼핵감산중복적Nogo-B기인cDNA적5'단서렬(the 5 ' terminal of Nago-B cDNA,Am-Nogo-B)급G-C함량위74％적부함CAG중복여CCG중복적Huntingtin기인cDNA적5'단서렬(the 5'terminal of huntingtin cDNA,Am-HTT)위례,재Sanger법측서체계중가입농도제도적첨채감,비교측서결과.결과 Am-Nogo-B여Am-HTT기인측서체계중최괄의적첨채감농도상차교대.첨채감농도재0.8～1.2 mol/L지간시,Am-Nogo-B측서결과질량최가.첨채감농도재1.6～2.4 mol/L지간시,Am-HTT기인적측서질량최호.결과구유량호적가중복성.결론 불동감기조성류형적고G-C함량DNA편단,즉사G-C함량접근,최괄의측서체계중첨채감농도불동.탐색출료괄의우증가부함삼핵감산천련중복적고G-C함량DNA편단측서변식도적체계,가작위유사DNA편단측서적삼고방법.
Objective To develop an optimal sequencing system which can improve the resolution of sequencing G-C 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 5' terminal of Huntingtin cDNA (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 G-C％ required different concentrations of betaine in the sequencing system due to base pair compositions.The sequencing system developed for improving the resolution of sequencing of G-C rich DNA with abundant trinucleotide repeats can be used as a reference for similar studies.
