细胞裂解法对单细胞PCR扩增效率的影响

目的研究细胞裂解法对单细胞PCR扩增效率的影响,寻求较好的细胞裂解方法.方法应用显微分离技术分离单个淋巴细胞,分别使用冻融变性和蛋白酶K裂解法处理单细胞,应用多重PCR同时扩增X染色体特异重复序列(DXZ1)和Y染色体特异重复序列(DYZ1)对单细胞进行性别诊断,比较其扩增效率.结果使用显微操作法获得80个男性淋巴细胞.采用冻融变性处理单细胞,40个淋巴细胞经多重PCR扩增后有32个产生DXZ1和DYZ1两条电泳带,正确率为80%.采用蛋白酶K裂解法,40个淋巴细胞经多重PCR扩增后均产生DXZ1和DYZ1两条电泳带,正确率为100%.两者的扩增效率有显著的差异性(χ2=6.81,P<0.01).结论 Rechitsky等的蛋白酶K裂解法比较简便,费时短,扩增效率高,适合细胞裂解及PCR扩增模板的制备.     

特纳综合征患者Y染色体序列的检测

目的观察特纳综合征患者Y染色体序列出现的频率。方法研究本院23位有特纳综合征且知情同意的患者,其中,细胞遗传学是通过培养外周血淋巴细胞进行染色体核型分析,每个病人分析100个核型。基因组DNA是通过提取外周血淋巴细胞DNA,通过聚合酶链式反应扩增基因序列DYZ1、DYZ3、ZFY和SRY。结果细胞遗传学分析显示其中9位病人(39.2%)是45,X的核型,14位病人(60.8%)是嵌合的核型。8.7%(2/23)的病人被发现有Y染色体序列。这个比率与之前报道的近似。在细胞遗传学分析中并没有发现有Y染色体片段,但是通过淋巴细胞DNA分析却发现有Y染色体特异性序列。结论PCR技术显示2位特纳综合征病人(8.7%)有Y染色体序列,而其细胞遗传学分析均存在标记染色体。     

PCR扩增孕妇外周血DNA进行胎儿性别诊断

采用密度梯度离心技术对40例孕妇外用血中胎儿细胞进行富集,并用PCR技术扩增了Y染色体特异重复序列,以产前基因诊断胎儿性别,结果38例胎儿性别诊断准确,证明此技术可作为无创伤性方法用于X连锁遗传病的产前诊断.     

PCR方法对单细胞SRY基因扩增效率的对比研究

目的探讨单细胞基因扩增时,巢式PCR和引物预扩增(PEP)-巢式PCR两种扩增方法对SRY基因脱扣的影响程度;并应用PEP-巢式PCR方法对单个卵裂球细胞进行SRY基因诊断。方法获取单个正常男女淋巴细胞,随机分为巢式PCR组和PEP-巢式PCR组。同时扩增SRY基因和ZP3基因位点。选用IVF-ET后冻存的4个胚胎,处理后获取单个卵裂球11个,用PEP-巢式PCR扩增,鉴定其性别。结果单个淋巴细胞经巢式PCR和PEP-巢式PCR方法扩增后,其基因扩增成功率分别为92.39%,98.91%;性别诊断正确率分别为86.00%,98.00%;SRY基因的脱扣率分别为16.67%,2.38%。两者有统计学检验均有显著性差异(P<0.05。对单个卵裂球应用PEP-巢式PCR方法进行SRY基因和ZP3基因扩增后,有3个胚胎的8个卵裂球被诊断为男性,而另外1个胚胎的3个卵裂球被诊断为女性。结论1.行单细胞基因扩增时,PCR扩增方法会影响等位基因脱扣的发生率。2.对性连锁遗传病进行植入前遗传学诊断时,采用PEP-巢式PCR方法扩增SRY基因和ZP3基因对单个细胞对进行性别鉴定时,可以有效地降低SRY基因脱扣的发生率,提高性别诊断的特异性和敏感性,能够用于单细胞的性别诊断,可用于性连锁遗传病的植入前遗传学诊断。     

46，XX男性患者血、尿、毛发、口腔上皮细胞DYZ1基因扩增研究

本文应用聚合酶链式反应(PCR)技术扩增了1例46,XX男性综合征患者的血、尿、毛发、口腔上皮细胞的Y染色体特异片段(DYZ1基因),结果均呈阳性。表明该患者存在有Y片段的移位。具体定位有待于进一步作染色体的原位杂交。     

羊水衍生X染色体的细胞与分子遗传学分析

目的 对1例孕中期胎儿46,X,der(X)行细胞与分子遗传学研究,并探讨其临床效应.方法 采用羊水细胞培养和G、C显带技术制备染色体,应用X染色体计数探针、Y染色体计数探针、Tel Xp/Yp三色荧光原位杂交技术(fluorescence in situ hybridization,FISH)进一步分析确定其核型.结果 衍生染色体为罕见的X/Y染色体的易位,其核型为:46,X,der(X)t(X;Y)(p22.3;q11.2).ish der(X)t(X;Y)(p22.3;q11.2)(X/Ypter-,DXZ1+,DYZ1+)mat.结论 FISH结合细胞遗传学检测可以查明衍生染色体的来源和性质,从而为产前诊断提供更全面准确的遗传学依据,并能预测胎儿发生畸形的风险及准确地判断预后.     

用荧光原位杂交和聚合酶链反应技术鉴定无精症患者标记染色体的来源

目的　研究无精症患者标记染色体的来源 ,对无精症患者进行明确的遗传学诊断。方法　应用双色荧光原位杂交 (fluorecence in situ hybridization,FISH)和聚合酶链反应 (polymerase chain reaction,PCR)技术对 2例无精症患者进行了分子细胞遗传学和分子遗传学检测。结果　确定了两例特发性无精症患者的标记染色体均来源于 Y染色体 ,确定其核型为 :4 6 ,X,·ish del(Y) (q11) (DYZ3+,DXZ1- )。结论　 FISH结合 PCR技术是鉴定标记染色体来源的又一非常重要方法     

宫颈管内滋养细胞进行染色体异常无创性产前诊断

目的 收集孕早期存在于妊娠妇女宫颈管内的脱落胎儿滋养细胞,采用定量荧光PCR(quantitative fluorescent-PCR,QF-PCR)技术复合扩增多个短串联重复序列(sllort tandem repeat,STR),建立一种染色体异常无创性产前诊断方法.方法 收集100例人工流产孕妇宫颈内口冲洗液,提取冲洗液中的DNA,分别针对21号、X、Y染色体上7个位点和13号、18号染色体上8个位点应用QF-PCR方法进行复合扩增检测并分析结果.对人流后的绒毛采用染色体核型分析确定染色体状态.结果 100例滋养细胞样本中,82例扩增成功,并准确检出样本的性别.15例检出核型正常,其他样本存在母体细胞污染.结论 经宫颈获取胎儿滋养细胞为孕早期无创性产前诊断提供了一个可能途径,但应采取更有效的措施富集胎儿滋养细胞,避免母体细胞污染.     

宫颈管内滋养细胞进行染色体异常无创性产前诊断

目的 收集孕早期存在于妊娠妇女宫颈管内的脱落胎儿滋养细胞,采用定量荧光PCR(quantitative fluorescent-PCR,QF-PCR)技术复合扩增多个短串联重复序列(sllort tandem repeat,STR),建立一种染色体异常无创性产前诊断方法.方法 收集100例人工流产孕妇宫颈内口冲洗液,提取冲洗液中的DNA,分别针对21号、X、Y染色体上7个位点和13号、18号染色体上8个位点应用QF-PCR方法进行复合扩增检测并分析结果.对人流后的绒毛采用染色体核型分析确定染色体状态.结果 100例滋养细胞样本中,82例扩增成功,并准确检出样本的性别.15例检出核型正常,其他样本存在母体细胞污染.结论 经宫颈获取胎儿滋养细胞为孕早期无创性产前诊断提供了一个可能途径,但应采取更有效的措施富集胎儿滋养细胞,避免母体细胞污染.     

宫颈管内滋养细胞进行染色体异常无创性产前诊断

目的 收集孕早期存在于妊娠妇女宫颈管内的脱落胎儿滋养细胞,采用定量荧光PCR(quantitative fluorescent-PCR,QF-PCR)技术复合扩增多个短串联重复序列(sllort tandem repeat,STR),建立一种染色体异常无创性产前诊断方法.方法 收集100例人工流产孕妇宫颈内口冲洗液,提取冲洗液中的DNA,分别针对21号、X、Y染色体上7个位点和13号、18号染色体上8个位点应用QF-PCR方法进行复合扩增检测并分析结果.对人流后的绒毛采用染色体核型分析确定染色体状态.结果 100例滋养细胞样本中,82例扩增成功,并准确检出样本的性别.15例检出核型正常,其他样本存在母体细胞污染.结论 经宫颈获取胎儿滋养细胞为孕早期无创性产前诊断提供了一个可能途径,但应采取更有效的措施富集胎儿滋养细胞,避免母体细胞污染.     

Sexing of human embryos and fetuses by fluorescent in situ hybridization (FISH) to paraffin-embedded tissues with sex chromosome-specific DNA probes

A fluorescent in situ hybridization (FISH) study was carried out on paraffin sections of human embryonic and fetal tissues with two DNA probes, DXZ1 and DYZ1 (Oncor), for X and Y chromosome-specific DNA sequences, respectively. The specificity of the DNA probes was confirmed on metaphase and interphase lymphocytes of healthy normal adult males. The paraffin blocks of the human embryonic and fetal tissues examined in the present study had been stored at room temperature for up to 5 years after fixation in 4% paraformaldehyde. All the seven embryos and five fetuses examined were successfully sexed by FISH. The cells from three embryos and four fetuses were positive for a hybridization signal with each of the DXZ1 and DYZ1 probes and they were classified as male. The cells from the remaining four embryos and one fetus were positive for two identical hybridization signals with the DXZ1 probe in a nucleus instead of the absence of the signal hybridized with DYZ1, indicating that their cells have two X chromosomes but no Y chromosomes. The FISH results for the five fetuses examined were consistent with their genital sex and/or gonadal histology. The FISH results were highly specific and no false positive or false negative results were obtained. Thus, the FISH technique has been shown to visualize specific DNAs in situ on paraffin sections and to be useful to determine the sex of fixed embryos and fetuses retrospectively. © 1994 Wiley-Liss, Inc.     

46,XY真两性畸形基因分析及发病机理初探

为了解４６，ＸＹ真两性畸形发病机理，采用多聚酶链式反应（ＰＣＲ）及核苷酸序列分析等手段，对４例４６，ＸＹ真两性畸形儿的ＳＲＹ基因及ＤＹＺ１序列进行了分析。４例患儿ＳＲＹ基因功能保守区均未发现基因突变。其中１例患儿ＰＣＲ扩增ＤＹＺ１序列未见特异扩增带，Ｓｏｕｔｈｅｒｎ杂交和细胞遗传学分析提示该患儿可能存在ＤＹＺ１序列的部分或全部缺失。对患儿父亲ＤＹＺ１序列分析的结果同正常男性进行了对照。综合有关报道，分析了ＳＲＹ基因及ＤＹＺ１序列在性别发育中的作用。     

孕妇血浆中胎儿DNA在产前诊断中的应用

目的 探讨孕妇血浆中胎儿DNA在无创性产前诊断中的应用。方法 应用酚—氯仿法抽提44名孕7～41周妇女血浆中胎儿DNA，通过PCR扩增胎儿Y染色体上DYZl位点，长度为149bp。结果 22名妊娠男性胎儿孕妇中全部出现DYZ1基因扩增条带，检出率为100．00％。22名妊娠女性胎儿孕妇中20例为阴性结果，两例假阳性结果。早、中、晚孕期性别符合率分别为88．89％、100．00％、96．55％，总符合率为95．45％。结论 使用酚—氯仿法抽提血浆中的DNA。提高模版的浓度和纯度进行PCR，增加了结果的准确性。提示母体血浆中游离的胎儿DNA可以作为无创性产前诊断的标本来源。     

The evaluation of gonosomal mosaics: lymphocyte interphase nuclei analyzed by FISH

The reliable evaluation of chromosomal mosaics is still considered to be difficult in clinical diagnosis if aberrant metaphases are only present at low frequencies. Classical cytogenetic findings cannot significantly exclude low mosaic levels, obviously, because of the relatively low number of analyzed metaphases. To study this problem, the number of gonosomes in lymphocyte interphase nuclei was determined by FISH (fluorescence in situ hybridization) application of two satellite DNA probes, DXZ1 and DYZ1. The results obtained with this method from lymphocytes of clinically and cytogenetically inconspicuous persons showed a high degree of reliability. The DNA probe yielded correct signals in more than 95% of the analyzed nuclei. Additionally, patients were examined who showed cytogenetically confirmed numerical gonosome aberrations. These results were compared with those obtained from the control group of inconspicuous patients and discussed with respect to the evaluation of gonosomal mosaics.     

A man who inherited his SRY gene and Leri-Weill dyschondrosteosis from his mother and neurofibromatosis type 1 from his father

We report on a man with neurofibromatosis type 1 (NF1) and Leri-Weill dyschondrosteosis (LWD). His father had NF1. His mother had LWD plus additional findings of Turner syndrome (TS): high arched palate, bicuspid aortic valve, aortic stenosis, and premature ovarian failure. The proband's karyotype was 46,X,dic(X;Y)(p22.3;p11.32). Despite having almost the same genetic constitution as 47,XXY Klinefelter syndrome, he was normally virilized, although slight elevation of serum gonadotropins indicated gonadal dysfunction. His mother's karyotype was mosaic 45,X[17 cells]/46,X,dic(X;Y)(p22.3;p11.32)[3 cells].ish dic(X;Y)(DXZ1 +,DYZ1 + ). The dic(X;Y) chromosome was also positive for Y markers PABY, SRY, and DYZ5, but negative for SHOX. The dic(X;Y) chromosome was also positive for X markers DXZ1 and a sequence < 300 kb from PABX, suggesting that the deletion encompassed only pseudoautosomal sequences. Replication studies indicated that the normal X and the dic(X;Y) were randomly inactivated in the proband's lymphocytes. LWD in the proband and his mother was explained by SHOX haploinsufficiency. The mother's female phenotype was most likely due to 45,X mosaicism. This family segregating Mendelian and chromosomal disorders illustrates extreme sex chromosome variation compatible with normal male and female sexual differentiation. The case also highlights the importance of karyotyping for differentiating LWD and TS, especially in patients with findings such as premature ovarian failure or aortic abnormalities not associated with isolated SHOX haploinsufficiency.     

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).     

应用分子生物学技术产前诊断Down综合征

目的 探讨应用PCR分子生物学方法产前诊断Down综合征（Down syndrome,DS)。方法 取产前诊断病例：羊水100例，绒毛16例。提取DNA，PCR扩增21号染色体的6个多态位点，电泳，膜转移，等位基因位点分析，诊断。结果 正常人为两种带型：杂合型显示两条带，纯合型一条带。Down综合征患者为三种带型：完全杂合型显示三条带，半杂合型两条带（信号增强的2：1带），纯合型一条带。100例羊水中2例阳性，16例绒毛标本中1例阳性，3例患者，至少有2个位点检出三个等 基因，患者为2个位点时，表现2：1带型；无一例正常检出三个等位基因。所有结果均与细胞染色体核型检查相符。结论 本分子生物学方法产前诊断DS简便、快速、可行，是一种值得推广的方法。     

A man who inherited his SRY gene and Leri‐Weill dyschondrosteosis from his mother and neurofibromatosis type 1 from his father

We report on a man with neurofibromatosis type 1 (NF1) and Leri‐Weill dyschondrosteosis (LWD). His father had NF1. His mother had LWD plus additional findings of Turner syndrome (TS): high arched palate, bicuspid aortic valve, aortic stenosis, and premature ovarian failure. The proband's karyotype was 46,X,dic(X;Y)(p22.3;p11.32). Despite having almost the same genetic constitution as 47,XXY Klinefelter syndrome, he was normally virilized, although slight elevation of serum gonadotropins indicated gonadal dysfunction. His mother's karyotype was mosaic 45,X[17 cells]/46,X,dic(X;Y)(p22.3;p11.32)[3 cells].ish dic(X;Y)(DXZ1 + ,DYZ1 + ). The dic(X;Y) chromosome was also positive for Y markers PABY, SRY, and DYZ5, but negative for SHOX. The dic(X;Y) chromosome was also positive for X markers DXZ1 and a sequence < 300 kb from PABX, suggesting that the deletion encompassed only pseudoautosomal sequences. Replication studies indicated that the normal X and the dic(X;Y) were randomly inactivated in the proband's lymphocytes. LWD in the proband and his mother was explained by SHOX haploinsufficiency. The mother's female phenotype was most likely due to 45,X mosaicism. This family segregating Mendelian and chromosomal disorders illustrates extreme sex chromosome variation compatible with normal male and female sexual differentiation. The case also highlights the importance of karyotyping for differentiating LWD and TS, especially in patients with findings such as premature ovarian failure or aortic abnormalities not associated with isolated SHOX haploinsufficiency. © 2001 Wiley‐Liss, Inc.     

胎鼠性别决定区蛋白基因快速检测（英文）

目的：为了将雄性胎鼠组织细胞移植进入雌性受体鼠，Y染色体特异的性别决定区蛋白基因（sex determining region protein, Sry）作为常用的遗传标志需要在细胞分离前快速检测|为了检测孕14 d胎鼠的Sry基因，我们建立了一种快速的PCR方法。基于C57BL/6J小鼠Sry基因序列，我们设计了特异检测引物，并优化了PCR的反应条件包括引物和循环参数等。方法和结果：模板的制备时间约30 min。取约1 mg胚胎组织，以10 mmol/L Tris 10 mmol/L EDTA pH 8.0 缓冲液洗涤2次，然后以200 μL裂解液（20 mg/L proteinase K, 0.5% NP-40, and 0.05% Tween 40）悬浮，再在60℃孵育15 min以消化蛋白，95℃以上5 min灭活蛋白酶K。取5-10 μL作为模板。PCR反应总体积为50 μL，使用两套引物同时扩增，一套扩增Sry目的基因片段，另一套扩增IL-3基因片段作为内对照。扩增时间为1.5 h。扩增产物10-15 μL采用2.5%-3%的琼脂糖凝胶在12-15 V/cm的电压下电泳。10 min即可观察电泳结果。根据引物设计判断结果，Sry目的基因片段为444 bp、IL-3基因片段为649 bp。操作约10个胚胎总时间小于3.5 h。PCR扩增的特异性与特异探针荧光原位杂交结果一致。结论：该PCR方法为检测胎鼠性别决定区蛋白基因的快速、简单、敏感及准确的方法。