Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments
Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments
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DOI：
10.1111/j.1462-2920.2005.00779.x
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发表时间：
2005-07-01
影响因子：
5.1
通讯作者：
Saiz-Jimenez, C
中科院分区：
文献类型：
--
作者：
Gonzalez, JM;Portillo, MC;Saiz-Jimenez, C
自然生物多样性的微生物评估通常通过聚合酶链反应（PCR）扩增来实现。由于PCR抑制剂的存在或特定序列的拷贝数低，来自天然样品的脱氧核糖核酸（DNA）序列通常难以扩增。在这项研究中，我们提出了一个非特异性的预扩增程序，以克服抑制剂的存在，并增加拷贝数之前进行标准扩增PCR。预PCR步骤通过多重置换扩增（MDA）技术在等温全基因组扩增反应中使用随机六聚体作为引发寡核苷酸和Phi 29 DNA聚合酶进行。使用特异性引发寡核苷酸的聚合酶链反应扩增允许在从复杂环境样品的预扩增反应之后选择感兴趣的序列。该程序（MDA-PCR）已被测试的天然微生物群落从地下环境和实验室组装已知的细菌物种，在这两种情况下，针对小亚基核糖体RNA基因序列。来自自然群落的结果显示使用本研究中提出的两步方案成功扩增，而标准的直接PCR扩增没有扩增产物。从实验室集合的扩增的两步提出的协议是成功的细菌浓度>= 10倍低于标准PCR。在不同浓度的富里酸（土壤腐殖酸部分）的存在下进行扩增的MDA-PCR协议产生的PCR产物的浓度超过10倍，比标准的PCR扩增的富里酸。所提出的程序（MDA-PCR）打开了检测在非常低的拷贝数的序列表示的可能性，与微小的样品，以及减少对PCR扩增的一些抑制性物质的负面影响，通常在环境样品中发现。
Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and Phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.