Quantitative analysis of microchimerism with Y-chromosome-specific PCR in canine small bowel transplantation.
Quantitative analysis of microchimerism with Y-chromosome-specific PCR in canine small bowel transplantation.
复制标题
犬小肠移植中 Y 染色体特异性 PCR 的微嵌合定量分析。
DOI：
10.1016/s0041-1345(00)01223-9
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发表时间：
2000
影响因子：
0.9
通讯作者：
Murase,N
中科院分区：
文献类型：
--
作者：
Okuda,T;Ichikawa,N;Zhu,Y;Chun,HJ;Demestris,AJ;Nalesnik,MA;Rudert,B;Trucco,M;Starzl,TE;Murase,N
方法用人源引物SRY-1F和SRY-2 R，2对雄性犬淋巴细胞进行PCR扩增，获得犬Y染色体PCR引物5′-CGTCAGACGACCCATGAA-3′和5′-CTCGGTGCATGGC CTGTA-3′（产物大小为188 bp）。在含有1.25 U Taq DNA聚合酶的50 μL总反应混合物中使用1.5 μg基因组DNA进行PCR。0.25μmol/L引物、0.2 mmol/L dNTP和10× PCR缓冲液（500 mmol KCl、20 mmol/L MgCl 2、100 mmol/L tris HCl）。0.1%明胶，调节至pH 8.4）。本研究中检测的可变PCR条件包括：变性; 94 - 95 ℃ 1分钟;退火; 55 - 62 ℃ 45 - 60秒;延伸：72 ℃ 45 - 60秒，使用DNA热循环仪480（Perkin Elmer Cetus，Norwalk，Conn）进行25 - 35个循环. PCR扩增后。将DNA转移到尼龙膜上，用32 P标记的狗SRY探针进行Southern印迹。暴露3小时后，如前所述通过PhosphorImager（Molecular Dynamics，桑尼维尔，加利福尼亚州）测量放射性。3.按照先前描述的程序在狗中进行小肠移植。4雄性和雌性成年猎犬（哈兰斯普拉格道利.印第安纳波利斯。结果从正常公、母犬外周血淋巴细胞中提取DNA，按1：0 ~ 1：10 6的比例混合，经变性、94 ℃退火1 min、60 ℃延伸1 min、100 ℃退火1 min、100 ℃延伸1 min、100 min、100 72 ℃ 1分钟，33个循环，第一次变性; 94 ℃ 3分钟，最后一次延伸; 72 ℃ 8分钟。在此条件下，具有已知雄性DNA浓度的样品的Southern杂交后的放射性根据雄性DNA浓度显示出几乎线性增加。雄性DNA浓度与放射性之间存在明确的相关性，并创建了用于半定量雄性DNA浓度的标准曲线。可以检测到低至0.0001%的雄性DNA浓度。
MethodsDog SRY sequence was cloned by PCR amplification of male dog lymphocytes using human primers; SRY-1F and SRY-2R, 2 and the primer set for the dog Y-chromosome PCR was obtained; 5′-CGTCAGACGACCCATGAA-3′ and 5′-CTCGGTGCATGGC CTGTA-3′(product size; 188 bps). PCR was performed with 1.5 μg genomic DNA in a 50 μL total reaction mixture containing 1.25 U Taq DNA polymerase. 0.25 μmol/L primers, 0.2 mmol/L dNTP, and 10× PCR buffer (500 mmol KC1, 20 mmol/L MgCl 2, 100 mmol/L tris HC1. 0.1% gelatin, adjusted to pH 8.4). Variable PCR conditions examined in this study included: denature; 94 to 95 C for 1 minute, annealing; 55 to 62 C for 45 to 60 seconds and extension: 72 C for 45 to 60 seconds with 25 to 35 cycles using DNA Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, Conn). After PCR amplication. DNAs were transferred onto nylon membranes, and southern blotting was performed using 32 P-labeled probe for dog SRY. After 3-hour exposure, radioactivity was measured by PhosphorImager (Molecular Dynamics, Sunnyvale, Calif) as previously described. 3 Small bowel transplantation was performed in dogs according to procedures previously described. 4 The male and female adult hound dogs (Harlan Sprague Dawley. Indianapolis. In) weighing 21 to 23 kg were used as donors and recipients, respectively.ResultsDNAs were prepared from peripheral blood lymphocytes of normal male and female dogs, and mixed at various ratios ranging from 1: 0 to 1: 10 6 Using these samples, appropriate PCR products were gained under denature; 94 C for 1 minute annealing; 60 C for 1 minute and extension; 72 C for 1 minute with 33 cycles with first denature; 94 C for 3 minutes, last extension; 72 C for 8 minutes. Under this condition, radioactivities after southern hybridization of samples with known male DNA concentrations showed nearly linear increase according to male DNA concentration. Clear correlation between male DNA concentration and radioactivity was achieved, and a standard curve was created for semiquantitation of male DNA concentration. It was possible to detect male DNA concentration as low as 0.0001%.
影响因子：
4.3
作者：
H. Sambrook
通讯作者：
H. Sambrook