Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO)
标记的高度简并引物(THDP)-PCR用于基于含铜膜结合型单加氧酶(CuMMO)的甲烷和氨氧化细菌的菌群分析
发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2354 浏览次数: 8204
评审: Dennis NürnbergPalash Kanti DuttaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2016
Abstract
We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This strategy remarkably increases the divergence of CuMMO gene amplicons while maintaining PCR efficiency without obvious amplification bias. This THDP-PCR strategy can be extended to other functional gene-based community analysis with design of new highly degenerate primer covering target functional gene sequences.
Keywords: Tagged highly degenerate primer (标记的高度简并引物)
Two-step PCR (两步法PCR)
CuMMO (CuMMO)
Background
Gene types in CuMMO superfamily are divergent and existent primer sets can only cover some CuMMO types (Tuomivirta et al., 2009). To cover the divergent types of genes in the CuMMO superfamily, highly degenerate primers are inevitable, but previous strategies using highly degenerate primers have limitations when applied to environmental samples, like low PCR efficiency or non-specific amplification (Ledeker and De Long, 2013). We recently used a two-step PCR strategy with tagged highly degenerate primers, designated THDP-PCR, to amplify a wide range of genes in the CuMMO family with satisfactory PCR efficiency and no obvious amplification bias (Wang et al., 2017).
Materials and Reagents
- Pipette tips (20 μl, 200 μl, 1,000 μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 3521-HR , 3551-HR , 3101-05-HR )
- 1.5 ml Eppendorf tubes (Eppendorf, catalog number: T9661-1000EA )
- Strip PCR tubes and caps (Roche Diagnostics, catalog number: 1667009001 )
- PowerSoilTM DNA Isolation Kit (Mo Bio Laboratories, catalog number: 12888-100 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
- Premix Taq (Takara Bio, catalog number: R004A )
- dNTP mixture
- Tris-HCl (pH 8.3)
- Potassium chloride (KCl)
- Magnesium chloride (MgCl2)
- AxyPrepTM PCR Cleanup Kit (Corning, Axygen®, catalog number: AP-PCR-250 )
- Primers
Equipment
- Pipettes (0-10 μl, 10-100 μl, 100-1,000 μl) (Eppendorf, catalog numbers: k03030 , k03031 , k03032 )
- Vortex-genieTM 2 (Mo Bio Laboratories, model: Vortex-Genie® 2 )
- PICO 17 centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 )
- Automated thermal cycler TP600 (Takara Bio, model: TP600 )
Software
- Mothur (www.mothur.org)
- BLAST+ (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/)
- Megan5 (http://ab.inf.uni-tuebingen.de/software/megan5/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wang, J., Xia, F., Zeleke, J., Zou, B. and Quan, Z. (2017). Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO). Bio-protocol 7(12): e2354. DOI: 10.21769/BioProtoc.2354.
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分类
微生物学 > 群落分析 > THDP-PCR
分子生物学 > DNA > PCR
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