高GC含量FMR-1基因的PCR扩增

目的　建立一种简便、快速的脆性X综合征基因筛查法。方法　联合应用二甲基亚和甜菜碱扩增FMR- 1基因的三核苷酸重复序列。结果　本方法大大提高了FMR - 1基因的扩增效率。结论　该方法简便、快速、经济 ,可用于群体普查和门诊快速筛查     

脆性X综合征的筛查及基因诊断

目的：建立一种简便快速初步筛查脆性X综合征智力缺陷基因FMR－1突变的方法。方法：采用套式PCR技术对新生儿及婴幼儿的足跟血X染色体上基因FMR－1CGG重复序列进行扩增,通过以其拷贝数的鉴定筛查其突变型。结果：共筛查5200全新生儿和婴幼儿,查出1例男婴患者,其母亲是携带者。结论：套式PCR能简便快速地初筛出人群中携带者和可疑患者,对脆性X综合征的早期诊断和产前诊断有应用价值。     

应用聚合酶链反应-限制性片段长度多态技术快速检测中国耳聋人群基因突变热点

目的 通过筛查耳聋基因热点突变:GJB2基因的235delC、SLC26A4基因的IVS7-2A>G和线粒体12S rRNA(12S)基因1555 A>G达到快速诊断耳聋患者.方法 多重PCR扩增包括GJB2、SLC26A4及12S基因的3个片段,限制性片段长度多态分析是否存在相应位点的突变.结果 200例耳聋患者中,共检测出235delC纯合突变18例,杂合突变18例;IVS7-2A>G纯合突变2例,杂合突变13例;1555 A>G突变8例.检测结果均与测序结果相符合.3个热点突变基因的致病单体的检出率为21.7%,基因诊断率为14%.结论 应用聚合酶链反应-限制性片段长度多态技术检测耳聋患者的热点突变是一种快速、简便、高效、经济的耳聋致病基因筛查的方法.     

应用等位基因特异性扩增检测经典型苯丙酮尿症PAH基因第6外显子基因突变

目的建立快速简便、特异灵敏的鉴定PAH基因第6外显子c.611A〉G突变热点的基因诊断方法。方法针对突变频率较高的PAH基因第6外显子的突变热点c.611A〉G,设计等位基因特异性扩增（amplification refractory mutationsystem,ARMS）的特异引物,对山西省已经临床确诊的70例经典型PKU患儿、c.611A〉G突变患儿的家长及50例正常儿童进行第6外显子扩增,扩增产物测序验证。结果在受检的山西省患儿中,PAH基因第6外显子c.611A〉G突变位点ARMS检测结果和测序结果完全相符。结论 ARMS技术操作简便,重复性和稳定性好,可作为PAH基因热点突变的快速灵敏检测方法。     

不明原因智力低下儿童脆性X综合征的筛查和基因诊断

目的建立一种快速分析脆性X综合征智力低下基因1(Fragile X mental retardation gene 1,FMR-1)突变的方法,对不明原因智力低下儿童进行脆性X综合征的筛查和诊断。方法应用7-deza-dGTP的PCR法一次性扩增FMR-1基因的(CGG)n的重复区,检测CGGn的重复序列的大小判断FMR-1基因状态(正常、突变前、突变后),对脆性X综合征可疑患儿快速筛查。结果在101例不明原因的先天性智力低下惠儿中,我们发现脆性X综合征患儿13例(男性10例,女性3例)。结论采用7-deza-dGTP扩增GC富集区的PCR法可对高危患儿进行快速筛查,确定携带者和患者。     

应用PCR技术对β地贫症进行基因诊断

本文应用DNA体外多聚酶链反应基因扩增新技术,结合斑点杂交,检测基因点突变的直接、快速、简便方法,对4例受试者进行了β地中海贫血症的基因分析和产前基因诊断。     

PCR-LIS-SSCP快速分析非缺失型α-地中海贫血点突变

目的建立一种简便快速的筛查非缺失型α－地中海贫血点突变的ＳＳＣＰ分析方法。方法在选择性扩增α２珠蛋白基因的基础上,巢式ＰＣＲ扩增突变热点区域,低离子强度（ＬＩＳ）条件下将ＰＣＲ产物热变性,用非变性胶电泳分析其ＳＳＣＰ。结果ＬＩＳ条件下处理样品,ＰＣＲ产物变性效率明显提高,电泳结果可检出区别于野生型对照的３种含基因突变的电泳带型,且电泳时间仅需３小时。结论ＰＣＲ－ＬＩＳ－ＳＳ－ＣＰ可用于快速筛查非缺失型α－地中海贫血点突变。     

应用PCR快速筛查脆性X综合征患儿

目的应用PCR快速筛查脆性X综合征患儿。方法采用PCR和聚丙烯酰胺凝胶电泳技术,对24例不明原因智力低下患儿的脆性X基因（CGG）n重复序列进行检测。结果在24例不明原因智力低下患儿中,筛查出1例脆性X综合征患者。结沦采用PCR技术扩增脆性X基因的（CGG）n重复序列,可对脆性X综合征患者进行快速筛查。     

反向点杂交快速诊断非缺失型α-地中海贫血

目的 建立一种简便快速筛查非缺失型α—地中海贫血点突变的反向点杂交(reverse dot blot,RDB)技术。方法 将生物素直接标记在引物上,选择性扩增人α2珠蛋白基因,将PCR产物与固化在膜条上的寡核苷酸探针杂交,洗膜、显色后分析结果。结果 采用生物素标记的引物：Bio—C1和Bio—C3可选择性扩增α2珠蛋白基因,PCR产物长度为1085bp,该标记片段与固相探针构成的反向点杂交检测体系,可以分辨出中国人群中已知的6种非缺失型α—地中海贫血点突变。结论 该反向点杂交检测体系无需巢式PCR扩增,一步选择性扩增的α2珠蛋白基因产物即可用于杂交;无需进行检测标记物(如生物素)反应,而将生物素直接标记在引物上;无需不同的洗膜条件,故较已报道的同类方法更为简单易行。RDB技术可用于快速诊断中国人非缺失型α—地中海贫血点突变。     

载脂蛋白E基因PCR-RFLP分析方法的建立

目的为研究新疆地区老龄人群载脂蛋白E(ApoE)基因多态性与阿尔茨海默病发病之间的关系而建立一种快速、简便、敏感的对ApoE基因进行多态性分析的方法.方法从抗凝血中提取人全基因组DNA,聚合酶链式反应(PCR)高效扩增ApoE基因,酶切后进行8%非变性聚丙烯酰胺凝胶电泳,凝胶银染色后进行ApoE基因型分析.结果限制性内切酶酶切图谱证实可特异性对ApoE基因进行分型,PCR-RFLP方法在ApoE基因型检测中得到成功建立.     

Molecular screening for fragile X syndrome in mentally handicapped children in Korea.

Fragile X syndrome is one of the most common forms of inherited mental retardation and is caused by the expansion of the CGG trinucleotide repeats in the FMR-1 gene. This study was aimed to facilitate the molecular screening of fragile X syndrome in Korean children with mental retardation of unknown etiology. The subjects were tested by Expand Long Template PCR system in the presence of 7-deaza-dGTP, and then by Southern blot analysis. The PCR method provided rapid and reliable results for the identification of fragile X negative and positive patients. One hundred one mentally retarded children (78 males and 23 females) were screened by PCR amplification, which detected only one abnormal sample. The PCR-positive case was confirmed by the CGG repeat expansion on Southern blot analysis with a positive cytogenetic result. In conclusion, Expand Long Template PCR may be used as the first screening test for detecting the fragile X syndrome.     

用RT-PCR技术分析脆性X男性患者的FMR-1基因的表达

目的为探讨脆性Ｘ男性患者与ＦＭＲ－１基因表达之间的关系，从而建立一种快速、简单且适合于筛查群体中男性患者的方法。方法采用ＲＴ－ＰＣＲ技术，对两个经细胞遗传学方法证实的脆性Ｘ家系进行检测。结果两个脆性Ｘ家系中的男性患者均无ＦＭＲ－１基因的表达，其父母均有ＦＭＲ－１基因表达，所有被检个体均有内对照基因ＨＰＲＴ的表达。结论ＦＭＲ－１基因表达缺失与脆性Ｘ男性患者的发病有一定关系。这一方法的进一步研究，将使脆性Ｘ男性患者的诊断方法更加简单、快速，有可能用于大规模群体筛查。     

Slowdown PCR法产前快速筛查脆性X综合征

目的研究产前快速筛查脆性X综合征的基因检测方法。方法收集临床疑似脆性X综合征的男性智力低下患儿病例,采集相应患儿外周血,同时收集需要进行胎儿脆性X综合征产前筛查和诊断的孕妇病例,在孕20w收集孕妇的羊水。通过slowdownPCR方法检测FMR1基因(CGG)n三核苷酸重复顺序基因组,并采用测序方法进行验证。结果共检测疑似病例30例,通过slowdown PCR方法简化了检测步骤,缩短了检测时间,整个检测过程缩短至2h,检测PCR产物片段与测序结果完全吻合。结论采用Slowdown PCR方法替代传统普通的PCR,提高了基因扩增效率,解决了由于基因CG含量过高导致PCR扩增困难和稳定性差的技术难题。     

Expand Long PCR for fragile X mutation detection

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)_5–3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.     

Fragile X syndrome carrier screening in the prenatal genetic counseling setting.

PURPOSE: To document our experience with fragile X carrier screening. METHODS: In this study, 29,103 women with no known or suspected family history of fragile X syndrome were offered fragile X carrier screening during their prenatal genetic counseling visit. Screening acceptance was analyzed by referral indication, carrier frequencies documented, and prenatal outcome data presented. RESULTS: Overall, 7.9% accepted carrier screening. The premutation frequency was 1 in 382, and the intermediate allele frequency was 1 in 143. CONCLUSIONS: Fragile X screening is a desirable option for some women seeking prenatal genetic counseling and should be made available to this population.     

A systematic review of population screening for fragile X syndrome

PurposeTo conduct a systematic review of literature regarding population-based screening for fragile X syndrome in newborns and women of reproductive age, either before or during pregnancy.MethodsSeven electronic databases were searched for English language studies published between January 1991 and November 2009. Data extraction was performed for all included studies. Results were synthesized using a narrative approach.ResultsOne article that examined offering newborn screening for fragile X syndrome and 10 that examined the offer of fragile X syndrome screening to women of reproductive age were identified. Two of these articles also addressed psychosocial aspects of population screening for fragile X syndrome such as attitudes to screening and experiences of screening, and a further nine addressed these issues alone. Studies exploring psychosocial issues demonstrated challenges for counseling arising from a lack of awareness or personal experience with fragile X syndrome in the general population.ConclusionsTargeted counseling and educational strategies will be essential to support women from the general population. It is crucial that future studies offering screening for fragile X syndrome explore a range of psychosocial aspects in addition to looking at uptake of testing and mutation frequency.     

非同位素PCR及Southern印迹杂交分析检测脆性X综合征FMR-1基因突变

目的建立一种非同位素的检测脆性X综合征智力缺陷基因(FMR-1)突变的方法.方法采用PCR技术结合Southern 印迹杂交技术对FMR-1基因进行分析,进而确定其突变类型.结果共对190例样本进行了检测,其中2例为女性前突变携带者,1例为女性全突变患者.结论非同位素PCR方法可以简便、安全、可靠地检测FMR-1基因中(CGG)n 重复拷贝数,可作为临床上对脆性X综合征的筛查的首选方法;而非同位素Southern印迹杂交可对结果不确定者进一步进行检测.