PCR-aided DNaseI footprinting of single copy gene sequences in permeabilized cells.
PCR-aided DNaseI footprinting of single copy gene sequences in permeabilized cells.
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透化细胞中单拷贝基因序列的 PCR 辅助 DNaseI 足迹。
DOI：
10.1093/nar/18.19.5902
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发表时间：
1990
影响因子：
14.9
通讯作者：
Riggs,AD
中科院分区：
文献类型：
--
作者：
Tanguay,RL;Pfeifer,GP;Riggs,AD
结扎介导PCR （LMPCR）已被用于体内二甲基硫酸盐（DMS）足迹（1,2）和基因组测序(3)。LMPCR的基因组测序使用连接器连接步骤将共同的寡核苷酸序列引入DNA末端。这使得所有含有连接子的分子都可以通过使用连接子特异性引物和基因特异性引物进行PCR扩增。LMPCR扩增提供了分析相对少量DNA中的单拷贝基因所需的敏感性和特异性。连接体的成功连接需要基因组DNA片段上的5'磷酸基团，这是DNA i切割提供的一个特征。我们在这里报道，可以对渗透细胞上的单拷贝基因进行DNasel足迹。Y162-1IC是一种含有活性人类X染色体的中国仓鼠人杂交细胞系(4)。按照Zhang和Gralla(5)的描述，用0.5 mg/ml溶卵磷脂处理单层细胞（4 × 106个细胞），在37℃下处理1分钟，制备渗透性细胞。用25 ~ 100吉/毫升的DNaseI在室温下处理10分钟(5)，然后分离DNA(3)。从Y162-I1C细胞中纯化的DNA，通过DNaseI酶切（2jg DNA， 0.1至0.2 tg/ml DNaseI，室温下酶切2min）获得对照。含有人磷酸甘油酸激酶-1 （pgk-1）基因序列的DNA片段通过LMPCR特异性扩增(3)。直接用LMPCR扩增DNaseI产生的片段，扩增效率低，背景高。当基因组DNA片段的3'OH基团首先通过添加双脱氧核苷酸阻断时，结果显着改善。DNA处理后，将DNA（图10,50 tl）变性，与5单位序列酶2.0 (USB)， 5个tsM ddNTPs在40 mM Tris-HCl， pH 7.7, 25 mM NaCl， 6.8 mM MgCl2中在45℃下孵育20分钟，然后再次变性，与30单位末端转移酶（BRL）在37℃下孵育30分钟，相同的反应混合物中添加200 mM羧酸钾，pH 7.0, 1 mM 2-巯基乙醇。经苯酚/氯仿萃取和选择性乙醇沉淀后，按(3)所述进行LMPCR处理。图1显示了体内DNA的消化模式与裸基因组DNA的消化模式。所示区域包括转录起始位点和pgk- 1上游序列(6)。与裸DNA相比，通透化细胞显示出几个不同的DNA敏感带（图1，封闭符号）和保护带（图1，开放符号）。在体内保护免受DNaseI攻击的核苷酸位于两个相邻的SPI一致序列（nt.-40至65）中，先前显示在体内保护免受
Ligation mediated PCR (LMPCR) has beenused for in vivo dimethylsulfate (DMS) footprinting (1, 2) and genomic sequencing (3). Genomic sequencing by LMPCR uses a linker-ligation step to introduce a common oligonucleotide sequence to DNA ends. This enables all linker-containing moleculesto be PCR amplified by use of a linker-specific primer along with a gene-specific primer. LMPCR amplification gives the sensitivity and specificity necessary to analyze single copy genes in relatively small amounts of DNA. Successful ligation of the linker rerquires a 5'phosphate group on the genomic DNA fragments, a feature provided by DNaseI cleavage. We report here that DNasel footprinting can be done for single copy genes on permeabilized cells. Y162-1IC is a Chinese hamster human hybrid cell line containing an active human X chromosome (4). Permeabilized cells were prepared by treating cell monolayers (4x 106 cells) with 0.5 mg/ml lysolecithin for 1 min at 37 C as described by Zhang and Gralla (5). These cells were treated with DNaseI (25 to 100 jig/ml) at room temperature for 10 min (5) and DNA then isolated (3). Controls were obtained by DNasel digestion (2 jg of DNA with 0.1 to 0.2, tg/ml DNaseI for 2 min at room temperature) of purified DNA from Y162-I1C cells. DNA fragments containing human phosphoglycerate kinase-l (pgk-1) gene sequences were specifically amplified by LMPCR (3). Direct LMPCR amplification of DNaseI generated fragments resulted in a low amplification efficiency and high background. Results were considerably improved when the 3'OH groups of genomic DNA fragments were first blocked by addition of a dideoxynucleotide. After DNaseItreatment, DNA (10 fig in 50 tl) was denatured and incubated with 5 units of Sequenase 2.0 (USB), 5 tsM ddNTPs in 40 mM Tris-HCl, pH 7.7, 25 mM NaCl, 6.8 mM MgCl2 for 20 min at 45 C. The DNA was then denatured again and incubated with 30 units terminal transferase (BRL) at 37 C for 30 min in the same reaction mixture supplemented to 200 mM potassium cacodylate, pH 7.0, 1 mM 2-mercaptoethanol. After phenol/chloroform extraction and selective ethanol precipitation, the samples were processed for LMPCR as described (3). Figure 1 shows in vivo DNaseI digestion patterns compared to the digestion of naked genomic DNA. The region shown includes the transcription start site and upstream sequences ofpgk-l (6). When compared to naked DNA, permeabilized cells show several distinct DNaseI hypersensitive bands (Figure 1, closed symbols) and protected bands (Figure 1, open symbols). The nucleotides protected in vivo from DNaseI attack are located within two adjacent SPI consensus sequences (nt.-40 to-65) previously shown to be protected in vivo from
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人 X 连锁 3-磷酸甘油酸激酶基因启动子区域的序列。
影响因子：
3.5
作者：
J. Singer;Douglas H. Keith;Kenzaburo Tani;Robert L. Simmer;Louise Shively;Susan Lindsay;Akira Yoshida;Arthur D. Riggs
通讯作者：
Arthur D. Riggs
影响因子：
56.9
作者：
PFEIFER, GP;STEIGERWALD, SD;RIGGS, AD
通讯作者：
RIGGS, AD
影响因子：
56.9
作者：
MUELLER, PR;WOLD, B
通讯作者：
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影响因子：
10.5
作者：
L. Zhang;J. Gralla
通讯作者：
J. Gralla