Heteroduplex resolution using T7 endonuclease I in microbial community analyses.
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.
Lowell
J L
JL
Department of Microbiology, Colorado State University, Fort Collins 80523-1677, USA.
Klein
D A
DA
D016477:Artifacts; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004784:Environmental Monitoring; D005827:Genetics, Microbial / Q000379:methods*; D015698:Genomic Library; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D012988:Soil Microbiology
D004269:DNA, Bacterial; D004271:DNA, Fungal; D009692:Nucleic Acid Heteroduplexes; D012337:RNA, Ribosomal, 18S; D003850:Deoxyribonuclease I