e49 Nucleic Acids Research, 2005, Vol. 33, No. 5 PAGE 4 OF 7 Figure 2. Fluorescence microscopy image of E.coli containing pGRFP with cloned BAC library. The green cells are GFP+/DsRed and contain plasmids with successfully inserted fragments from the library. The orange colored cells are GFP+/DsRed+ and contain native pGRFP vector without inserts. This approach was validated by subcloning a sea urchin BAC library into pGRFP. Figure 2 shows an image of ﬂuor- escence from transformed E.coli excited by 450–490 nm light. The cells containing native pGRFP vector appear orange, because they express both GFP and DsRed. The insert- containing cells are readily recognized by their green color. There is little ambiguity about the presence or absence of inserts in each cell in the image. Cells were further analyzed using ﬂow cytometry. Figure 3B and C show bivariate dot plots of DsRed versus GFP ﬂuores- cence for each analyzed sample. Native pGRFP vector yields cells with a high DsRed to GFP ratio (Figure 3B). When foreign DNA is cloned into pGRFP, a second population of cells appear that does not have red ﬂuorescence (Figure 3C). This population is equivalent to the green cells shown in Figure 2 and represents clones with successfully integrated inserts. A large separation in the two populations is observed by the ﬂow cytometer according to the DsRed/GFP ﬂuores- cence ratio resulting in a high degree of certainty when discriminating between them. Reliability of sorting assessed by PCR amplification Cloned DNA inserts were PCR ampliﬁed from 46 single sorted cells (see Figure 4). All the 46 PCR products indicate the presence of cloned inserts. A total of 39 clones had inserts in the 1.5–3 kb size range of the original library, while 7 clones had inserts in the 400–1500 bp range. This experiment demon- strated that the GFP+/DsRed phenotype has a 100% predict- ive value in detecting the presence of inserts. We estimated the initial fraction of insert containing clones in culture by analyzing colonies grown from single sorted cells. In plates containing random cells deposited from the initial culture, a total of 588 colonies with 536 DsRed col- onies and 52 DsRed+ colonies were observed. In plates con- taining cells sorted for the GFP+/DsRed phenotype, 301 DsRed colonies and one DsRed+ colony were observed. This indicates a sorting enrichment factor of 29.2 for DsRed cells. Given this enrichment ratio, it is estimated that sorting from a culture with 90.0% GFP+/DsRed cells will yield nearly 100% cells with the correct phenotype. If sorting from a culture containing only 10.0% GFP+/DsRed cells, possibly as a result of inefﬁcient cloning, it is expected that 96.8% of sorted cells will have the desired phenotype. BAC sequencing We demonstrated the performance of the pGRFP dual- ﬂuorescence reporter system in addressing a biological ques- tion of interest. We sequenced a small portion of the genome of the sea urchin S.purpuratus as represented in BAC SU66E20. We subcloned this BAC, selected for insert- bearing clones by cell sorter, ampliﬁed plasmid DNA and sequenced the inserts. The average Phred20 score sequence read-length was 350 bases. Given the number of sequences used, coverage of 4.3X was achieved. Thirteen primary con- tigs were assembled that spanned 55 kb of sequence out of a total BAC length of 59 kb. Furthermore, sequenced clones were evenly spread throughout the SU66E20 BAC sequence. This level of successful assembly is expected, given the level of coverage and average sequence read-lengths. The results are similar to what would be achieved using traditional clone isolation and sequencing protocols. We analyzed the ﬁnal assembly data to evaluate the insert lengths of our ﬂow-sorted clones. The locations of the forward and reverse reads within the ﬁnal BAC assembly reveal the distance between these reads. We analyzed 113 clones that had both forward and reverse reads residing on either of the two largest contigs (13.0 and 7.3 kb in length). The mean insert length was 2.95 kb with a standard deviation of 860 bp. These insert sizes are well within the 2–4 kb range that was isolated during library construction. Six percent of the inserts were smaller than 2 kb in size. The insert sizes in the ﬁnal assembly approximate the insert sizes of the original library. DISCUSSION In this study, we report the development of the dual- ﬂuorescence approach to clone characterization and isolation. The ﬂow-cytometry data support our proposed mechanism of action of two linked ﬂuorescent proteins. Bacteria containing the pGRFP vector exhibit both green and red ﬂuorescence when excited with 488 nm light (Figure 3B). Integration of foreign DNA into cloning sites between GFP and DsRed res- ults in premature termination before DsRed is translated. This termination results in bacteria with primarily green ﬂuores- cence, represented as a second population of cells (Figure 3C). Despite the fact that bacteria have large cell-to-cell variations in ﬂuorescence, GFP acts as an internal control to interpret the level of DsRed ﬂuorescence. In this manner, two distinct popu- lations can be easily distinguished. The pGRFP vector performed very well in clone selection experiments. All the isolated GFP+/DsRed bacteria (100%) had inserts ampliﬁable using PCR. Furthermore, analysis of colonies grown from single sorted cells indicated that cell- sorters can enrich for GFP+/DsRed cells by at least a factor of 29.2. Given this enrichment factor, we estimate that cell- sorters could isolate cells containing inserts 96.8–100% of the