Universal amplification of DNA isolated from small regions of paraffin-embedded, formalin-fixed tissue.
Universal amplification of DNA isolated from small regions of paraffin-embedded, formalin-fixed tissue.
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从石蜡包埋、福尔马林固定组织的小区域中分离出的 DNA 进行通用扩增。
作者：
Stefan W. Faulkner;Don A. Leigh
从福尔马林固定的石蜡包埋的组织中分离的DNA的聚合酶链反应（PCR）分析可能是困难的，因为可用于扩增的模板DNA的固有质量差。福尔马林固定过程的结果是，DNA与蛋白质复合，并且经常被切割，产生相对较短的片段。这些样本通常DNA浓度低，质量差。随机失败率高，无法常规扩增大于650 bp的片段以及储存条件差，使得石蜡包埋的组织不是理想的DNA模板来源（2）。我们想用微卫星标记检测石蜡包埋组织切片中很小区域的特定细胞的杂合性缺失（洛）。为了从少量石蜡包埋的细胞中成功提取DNA，已经编写了各种方案（2，5），但是由于前面描述的困难，这些方案不适合基因组筛选项目。通用基因组扩增是一种克服与从石蜡包埋组织中分离的DNA相关的问题的方法。已经报道了几种用于生成合成基因组模板的方案，最值得注意的是Alu PCR（4）、简并寡核苷酸引物PCR（DOP-PCR）（8）和引物延伸预扩增（PEP）（9）。后者已被应用于实现单细胞的多重PCR检测，最近已被证明可用于扩展有限的DNA样品（1）。PEP方案的优势与PCR相反，它依赖于随机扩增，而不偏好特定序列。使用标准盐析程序从EDTA中收集的血液中制备对照基因组DNA，并通过荧光定量法进行定量（3）。通过以下程序从福尔马林固定的石蜡包埋的组织切片中分离DNA，所述组织切片的年龄、固定方法或固定时间未知。从苏木精和伊红染色切片中鉴定细胞区域，从单个相应的未染色10 μm载玻片切片上刮取选定细胞，并转移至500 μL PCR管中。刮擦材料的面积范围为约1至10 mm 2，取决于所选单元的数量。将样品在20 μL 1× PCR缓冲液II（Perkin-Elmer，Norwalk，CT，USA）中在石蜡油下加热至95 ℃，持续10 min，定期混合以帮助每个样品脱蜡。短暂冷却后，在65°C下用蛋白酶K（1000 μg/mL）消化样品90 min，然后在85°C下灭活酶15 min。然后将该溶液（10 μL）用作通用扩增的DNA模板。较大的样品通过氯仿提取，然后通过盐/异丙醇沉淀进行纯化。将DNA沉淀重悬于20 μL TE缓冲液（2 mM TrisHCl，0.2 mM EDTA，pH 8.0）中。使用PEP方案的修改产生通用扩增产物（9）。标准PEP反应在石蜡油中进行，体积为60 μL，含200 pmol随机12聚体寡核苷酸、5 U AmpliTaq DNA聚合酶（Perkin-Elmer）、200 μM各dNTP、5 mM MgCl 2、1× PCR缓冲液II和DNA模板。模板由从石蜡包埋组织切片中提取的纯化DNA、10 μL从石蜡包埋组织切片中解剖的细胞的蛋白酶K消化物或已知量的对照DNA组成。包括水空白作为每个扩增系列的污染检查。在
The polymerase chain reaction (PCR) analysis of DNA isolated from formalin-fixed, paraffin-embedded tissue can be difficult because of the inherently poor quality of template DNA available for amplification. As a consequence of the formalin fixation process, DNA is complexed with proteins and is often nicked, giving relatively short fragments. Such samples are often of low DNA concentration and poor quality. A high rate of random failure, the inability to routinely amplify fragments greater than 650 bp and poor storage make paraffin-embedded tissue a less than ideal source of DNA template (2). We wanted to assay specific cells from very small regions of paraffin-embedded tissue sections for loss of heterozygosity (LOH) using microsatellite markers. Various protocols have been written for the successful extraction of DNA from a small number of paraffinembedded cells (2,5), but these protocols were not amenable to genomic screening projects because of the difficulties previously described. Universal genomic amplification is a way to overcome the problems associated with DNA isolated from paraffin-embedded tissue. There have been several protocols reported for the generation of synthetic genomic template, the most notable being Alu PCR (4), degenerate oligonucleotide-primed PCR (DOP-PCR) (8) and primer extension pre-amplification (PEP) (9). The latter has been applied to achieve multiple PCR assays on single cells and has recently been proven useful for extending limited DNA samples (1). The strength of the PEP protocol is the opposite of PCR—it relies on random amplification without any preference for specific sequences. Control genomic DNA was prepared from blood collected in EDTA using standard salting out procedures and quantitated spectrophotometrically (3). DNA was isolated from formalin-fixed, paraffin-embedded tissue sections of unknown age, fixation method or fixation time by the following procedure. Regions of cells were identified from hematoxylin and eosin-stained sections, and the selected cells were scratched from a single corresponding, unstained 10-μm slide section and transferred to 500-μL PCR tubes. The area of the scratched material ranged from about 1 to 10 mm2, depending on the number of cells selected. Samples were heated to 95°C for 10 min under paraffin oil in 20 μL of 1× PCR Buffer II (Perkin-Elmer, Norwalk, CT, USA) with periodic mixing to aid de-waxing of each sample. After cooling briefly, samples were digested with proteinase K (1000 μg/mL) at 65°C for 90 min followed by enzyme inactivation at 85°C for 15 min. This solution (10 μL) was then used as DNA template for universal amplification. Larger samples were purified by chloroform extraction followed by salt/isopropyl alcohol precipitation. DNA pellets were resuspended in 20 μL TE buffer (2 mM TrisHCl, 0.2 mM EDTA, pH 8.0). Universal amplification products were generated using a modification of the PEP protocol (9). Standard PEP reactions were performed under paraffin oil in 60-μL volumes containing 200 pmol of random 12-mer oligonucleotides, 5 U AmpliTaq DNA Polymerase (Perkin-Elmer), 200 μM of each dNTP, 5 mM MgCl2, 1× PCR Buffer II and DNA template. Templates consisted of either purified DNA extracted from paraffin-embedded tissue sections, 10 μL of the proteinase K digest of cells dissected from paraffinembedded tissue sections or a known quantity of the control DNA. Water blanks were included as contamination checks for each amplification series. In