Metagenome microarray for screening of fosmid clones containing specific genes.
A critical step in the process of metagenome analysis is to screen for clones that contain specific genes among a large number of clones. To form one of the sequence-based screening tools of a metagenome library, we designed a format of microarray [metagenome microarray (MGA)] that is arrayed with fosmid library clone DNA samples on a glass slide. We evaluated the MGA using random prime labeled fluorescent probes prepared from PCR products of the target gene and found that we could obtain specific hybridization signals only for the fosmid clone that contained the target gene. We found that the detection limit of the MGA was c. 10 ng microL(-1) of fosmid clone DNA, and that the MGA-based hybridization was quantitative within a concentration range of 10-200 ng microL(-1) of fosmid clone DNA. We used the MGA successfully to identify two fosmid clones that contained 16S rRNA genes from a fosmid library from the sediment of the East Sea, Korea. In conclusion, we have demonstrated that the MGA can be used for screening for fosmid clones containing specific genes in a metagenome library, and that this technology has potential application as a high-throughput metagenome screening tool.
Park
Soo-Je
SJ
Department of Microbiology, Chungbuk National University, Heungduk-gu, Cheongju, Korea.
Kang
Cheol-Hee
CH
Chae
Jong-Chan
JC
Rhee
Sung-Keun
SK
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D020459:Genes, rRNA; D019015:Geologic Sediments / Q000382:microbiology; D007723:Korea; D046228:Microarray Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S