The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.

DOI link for The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.

The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.