247
Sea peR clone library exactly matched those from cultured species, all were specifically
related to cultured marine cyanobacteria, some at the near-species level. Most likely, no exact
matches were found because the data base of marine cyanobacterial reference sequences is still
Table I. Comparison of different approaches used for cloning rRNA genes from mixed microbial populations.
RIBOSOMAL coNA
LIBRARY
APVANTAGES
RIGOROUS LYSIS PROTOCOL
POSSIBLE
"ALL" RECOMBINANTS rDNA
CLONES
MAY SELECT "AC'nVE"
COMMUNITY MEMBERS
RIGOROUS LYSIS PROTOCOL
POSSIBLE
"ALL" RECOMBINANTS rDNA
CLONES
EXTREMELY SENSITIVE-NEED
VERY LOW AMOUNTS of DNA
CAN SELEcnvELY AMPUFY
SPECIFIC GROUPS
PISADVANTAGES
RNA PRONE to CHEMClAland
ENZYMAncDEGRADAnON
PROBLEMS In RECOVERY OF FULL
LENGTH eDNA
POTENnAL BIAS DUE to VARIABLE
rRNAICELL, and PRIMER
ANNEALING and CLONING STEPS
peR rONA
LIBRARY
POTENnAL FOR TAQ DNA POLYMERASE
ERROR
POSSIBIUTY of "SHUFFLE GENES" or
CHIMAERIC PCR PRODUCTS
EXTREMELY SENSITIVE-BACKGROUND
CONTAMINAnON A POTENnAL PROBLEM
POTENnAL SELEC'nVE BIAS DURING
PRIMER ANNEAUNG and AMPUFICAnON
"SHOTGUN" DNA
LIBRARY
UNBIASED BY PRIMER
EX'TENSIOfiLIAMPLlFlCAnON
STEP
LIBRARY EASILY STORED
POTENnAL for CHROMOSOME
'WALKING' ancllSOLAnoN of
OTHER GENES
NEED GENTLE LYSIS PROTOCOL
NEED LARGE AMOUNTS of DNA
POTENnAL SELEcnvE BIAS IN
ENDONUCLEASE DIGESTION, CLONING
AND CLONE IDENnFICAnON STEPS
LABOR INTENSIV!