中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2010年
9期
659-663
,共5页
郑巧娟%甘世锐%王柠%吴志英
鄭巧娟%甘世銳%王檸%吳誌英
정교연%감세예%왕저%오지영
脊髓小脑共济失调%三核苷酸重复%神经组织蛋白质类%核蛋白质类%克隆,分子%可重复性,结果
脊髓小腦共濟失調%三覈苷痠重複%神經組織蛋白質類%覈蛋白質類%剋隆,分子%可重複性,結果
척수소뇌공제실조%삼핵감산중복%신경조직단백질류%핵단백질류%극륭,분자%가중복성,결과
Spinocerebella ataxias%Trinucleotide repeats%Nerve tissue proteins%Nuclear proteins%Cloning,molecular%Reproducibility of results
目的 探讨克隆测序技术用于检测三核苷酸(CAG)重复次数的可靠性.方法 对1例临床确诊的遗传性脊髓小脑性共济失调1型(SCA1)患者,PCR法扩增ATXN1基因的CAG重复次数,8%变性聚丙烯酰胺凝胶电泳(DPAGE)分离PCR产物以协助确诊.2.5%琼脂糖凝胶电泳分离PCR产物的大小片段,割胶回收大小片段测序.另外将割胶回收的大片段进行TA克隆测序.结果 DPAGE提示大片段存在CAG的异常扩增,故该患者可确诊为SCA1.PCR产物大小片段直接测序结果为:小片段为26次CAG重复,大片段为47次重复.而将大片段克隆测序后,则有50、47、46、41、32、28、27、26、25及24次共10种不同的CAG重复次数,而且出现了CCG、CGG、CTG、CAA、TAT等序列变异现象.结论 克隆测序检测CAG重复次数会造成重复次数变异,且存在各种碱基变异.因此不宜单用TA克隆测序检测CAG重复次数及筛查碱基变异,需联合应用多种方法以提高结果的可靠性.
目的 探討剋隆測序技術用于檢測三覈苷痠(CAG)重複次數的可靠性.方法 對1例臨床確診的遺傳性脊髓小腦性共濟失調1型(SCA1)患者,PCR法擴增ATXN1基因的CAG重複次數,8%變性聚丙烯酰胺凝膠電泳(DPAGE)分離PCR產物以協助確診.2.5%瓊脂糖凝膠電泳分離PCR產物的大小片段,割膠迴收大小片段測序.另外將割膠迴收的大片段進行TA剋隆測序.結果 DPAGE提示大片段存在CAG的異常擴增,故該患者可確診為SCA1.PCR產物大小片段直接測序結果為:小片段為26次CAG重複,大片段為47次重複.而將大片段剋隆測序後,則有50、47、46、41、32、28、27、26、25及24次共10種不同的CAG重複次數,而且齣現瞭CCG、CGG、CTG、CAA、TAT等序列變異現象.結論 剋隆測序檢測CAG重複次數會造成重複次數變異,且存在各種堿基變異.因此不宜單用TA剋隆測序檢測CAG重複次數及篩查堿基變異,需聯閤應用多種方法以提高結果的可靠性.
목적 탐토극륭측서기술용우검측삼핵감산(CAG)중복차수적가고성.방법 대1례림상학진적유전성척수소뇌성공제실조1형(SCA1)환자,PCR법확증ATXN1기인적CAG중복차수,8%변성취병희선알응효전영(DPAGE)분리PCR산물이협조학진.2.5%경지당응효전영분리PCR산물적대소편단,할효회수대소편단측서.령외장할효회수적대편단진행TA극륭측서.결과 DPAGE제시대편단존재CAG적이상확증,고해환자가학진위SCA1.PCR산물대소편단직접측서결과위:소편단위26차CAG중복,대편단위47차중복.이장대편단극륭측서후,칙유50、47、46、41、32、28、27、26、25급24차공10충불동적CAG중복차수,이차출현료CCG、CGG、CTG、CAA、TAT등서렬변이현상.결론 극륭측서검측CAG중복차수회조성중복차수변이,차존재각충감기변이.인차불의단용TA극륭측서검측CAG중복차수급사사감기변이,수연합응용다충방법이제고결과적가고성.
Objective Cloning-sequencing is a common method to detect the number of trinucleotide repeats.The aim of the present study is to discuss its reliability.Methods One clinically diagnosed SCA1 patient was recruited in the study.The numbers of CAG repeats in ATXN1 gene were estimated via polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis (DPAGE).To verify accuracy of CAG numbers estimated, the PCR products were electrophoresed on a 2.5% agarose ge] and separated bands were excised for direct sequencing.Also, the longer separated band underwent cloning-sequencing using a TA cloning kit.Results The patient was identified as SCA1 by DPAGE.After direct sequencing, the numbers of CAG repeats were 26 and 47 in the shorter and longer bands, respectively.However, after cloning-sequencing of the longer band, there are 10 different numbers of CAG repeats, including 50, 47, 46, 41,32, 28, 27, 26, 25 and 24.Furthermore, there are other kinds of trinucleotide repeats, such as CCG, CGG, CTG, CAA and TAT scattered among the CAG repeats.Conclusions It is not reliable to identify the number of trinucleotide repeats by cloning-sequencing alone.To improve the reliability, it is better to combine cloning-sequencing with other methods.