Lowell J L, Klein D A
Department of Microbiology, Colorado State University, Fort Collins 80523-1677, USA.
Biotechniques. 2000 Apr;28(4):676-8, 680, 681. doi: 10.2144/00284st03.
Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.
使用分子技术（如PCR随后构建基因组文库）进行微生物群落分析，有助于更好地了解微生物群落。这在生态系统中尤为关键，因为在这些系统中，大多数现存微生物无法用传统技术培养。不幸的是，使用此类分子技术分析微生物群落结构存在问题，主要源于PCR产物的频繁形成。多模板PCR常常会出现诸如异源双链体形成等错误，这种情况是在从DNA混合群落中扩增特定基因的过程中产生的。基于本实验室的工作，在进行基因组文库构建之前，可以通过加入T7核酸内切酶I的消化步骤来解决异源双链体问题。在这里，从土壤群落DNA中扩增真菌的18S rDNA基因，并用T7核酸内切酶I进行消化，以在克隆之前解决PCR产物中存在的任何异源双链体。将这些样品与未接受T7核酸内切酶I处理的重复样品进行比较。在克隆之前，用10 U T7核酸内切酶I/微克DNA消化扩增的群落18S rDNA可消除异源双链体，只留下所需的克隆。未经T7核酸内切酶I处理时，在大约10%筛选的重组体中产生了异源双链体。增加这一步骤可以消除PCR产物中的异源双链体，并确保后续的基因组文库构建不受影响。
