PMID- 18467853
OWN - NLM
STAT- MEDLINE
DCOM- 20080806
LR  - 20191210
IS  - 1017-7825 (Print)
IS  - 1017-7825 (Linking)
VI  - 18
IP  - 4
DP  - 2008 Apr
TI  - Modified T-RFLP methods for taxonomic interpretation of T-RF.
PG  - 624-30
AB  - Terminal restriction fragment length polymorphism (TRFLP) is a method that has 
      been frequently used to survey the microbial diversity of environmental samples 
      and to monitor changes in microbial communities. T-RFLP is a highly sensitive and 
      reproducible procedure that combines a PCR with a labeled primer, restriction 
      digestion of the amplified DNA, and separation of the terminal restriction 
      fragment (T-RF). The reliable identification of T-RF requires the information of 
      nucleotide sequences as well as the size of T-RF. However, it is difficult to 
      obtain the information of nucleotide sequences because the T-RFs are fragmented 
      and lack a priming site of 3'-end for efficient cloning and sequence analysis. 
      Here, we improved on the T-RFLP method in order to analyze the nucleotide 
      sequences of the distinct TRFs. The first method is to selectively amplify the 
      portion of T-RF ligated with specific oligonucleotide adapters. In the second 
      method, the termini of T-RFs were tailed with deoxynucleotides using terminal 
      deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The 
      major T-RFs generated from reference strains and from T-RFLP profiles of 
      activated sludge samples were efficiently isolated and identified by using two 
      modified T-RFLP methods. These methods are less time consuming and 
      labor-intensive when compared with other methods. The T-RFLP method using TdT has 
      the advantages of being a simple process and having no limit of restriction 
      enzymes. Our results suggest that these methods could be useful tools for the 
      taxonomic interpretation of T-RFs.
FAU - Lee, Hyun-Kyung
AU  - Lee HK
AD  - Department of Microbiology, Chungbuk National University, Cheongju, Chungbuk 
      361-763, Korea.
FAU - Kim, Hye-Ryoung
AU  - Kim HR
FAU - Mengoni, Alessio
AU  - Mengoni A
FAU - Lee, Dong-Hun
AU  - Lee DH
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Korea (South)
TA  - J Microbiol Biotechnol
JT  - Journal of microbiology and biotechnology
JID - 9431852
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Bacterial)
RN  - 0 (Oligonucleotides)
RN  - EC 2.7.7.31 (DNA Nucleotidylexotransferase)
SB  - IM
MH  - Bacteria/*classification/genetics
MH  - DNA Nucleotidylexotransferase/metabolism
MH  - DNA Primers/genetics
MH  - DNA, Bacterial/genetics
MH  - Oligonucleotides/genetics
MH  - Polymerase Chain Reaction/*methods
MH  - *Polymorphism, Restriction Fragment Length
MH  - Restriction Mapping/*methods
EDAT- 2008/05/10 09:00
MHDA- 2008/08/07 09:00
CRDT- 2008/05/10 09:00
PHST- 2008/05/10 09:00 [pubmed]
PHST- 2008/08/07 09:00 [medline]
PHST- 2008/05/10 09:00 [entrez]
AID - 7229 [pii]
PST - ppublish
SO  - J Microbiol Biotechnol. 2008 Apr;18(4):624-30.