RapidScreen cDNA Library FAQs
1. How do Rapid-ScreenGäó Arrayed cDNA Library Panels work?
The Rapid-ScreenGäó cDNA Library Panels were designed for EST to "full-length" gene cloning and require just three sets of PCRs to identify the desired cDNA clone. The first PCR analysis is performed in a 96-well "Master Plate," where each well contains plasmid DNA from 5,000 clones. Having identified the positive well(s) by gel electrophoresis of the reaction products, the second PCR analysis is carried out in the corresponding 96-well "Sub-Plate(s)," which contains all 5,000 clones from the positive well of the Master Plate arrayed at only 50 clones per well. Since this plate contains glycerol stocks of E. coli instead of plasmid DNA, the cells can be diluted and plated out once a positive well(s) has been identified. Analysis of 96 single colonies by PCR leads to the identification of the positive clone from the Sub-Plate well.
To ensure that a clone detected in a specific well in the Master Plate will be represented in the appropriate Sub-Plate, the Sub- Plates were not derived by dilution of individual wells from the Master Plate (a process which may lose specific clones). Instead, the Sub-Plates were generated first and the wells from each Sub-Plate were then pooled to generate the Master Plate. To guarantee that clones from different wells in the Master Plate were each independently-derived; the Sub-Plates were generated with primary transformants that had not been amplified.
2. What was done to obtain large inserts?
A great deal of foresight has been incorporated into the development of these arrayed cDNA panels, so that they are user-friendly and cost-effective. Since they are intended for researchers searching for longer than average-sized cDNA clones, double-stranded cDNAs (synthesized from poly A+ RNA using oligo (dT) primers) were size-fractionated in a low-melt agarose gel and the various size-fractions were independently ligated to the vector to minimize competition for insertion by smaller cDNAs. The insert sizes of random clones from the various ligation reactions were then determined (Fig. 1). This allowed the Rapid-ScreenGäó libraries to be spiked with more clones from the large-sized fraction, thereby giving them a much larger average insert size (Table 1). In addition to helping find full-length clones, a large average insert size allows for screening of fewer members due to the general finding that longer transcripts tend to be less abundant in the cell. Since the cDNAs were directionally-cloned into the CMV expression vector pCMV6-XL4 (Fig. 2) a vector-derived 5' PCR primer can be used in conjunction with a gene-specific 3' reverse primer to identify the longest cDNA clone of interest (see Fig. 4).
3. Can the clones be expressed in mammalian cells?
Once a full-length clone has been isolated and sequenced, it can be directly transfected into mammalian cells for expression studies. The library plasmid, pCMV6-XL4, contains the SV40 origin of replication for propagation in mammalian cells. Moreover, the plasmid contains the CMV promoter, which will drive expression of the insert if it contains a translational start site. Finally, the human growth hormone polyadenylation signal is present to effect proper transcriptional termination.
4. Is a 96-well PCR necessary?
If it is not possible or desirable to perform 96 simultaneous PCRs, then pools of DNA from the wells in the Master Plate can be made. Using 5 -¦L of DNA from each well of the Master Plate, twelve pools (40,000 clones/pool) of DNA can be made from columns 1 through 12, and/or eight pools (60,000 clones/pool) of DNA can be made from rows A through H. These 20 pools can be analyzed by PCR using two gene-specific primers. In the example shown in Fig. 3, there is a single clone in this library and it is located in well B6 of the Master Plate (the last lane is a positive control). The one caveat to this approach is that since there are 40,000-60,000 independent clones per pool, rather than only 5,000 from an individual well, both gene-specific primers must be highly specific. The pooling technique is most informative when working with a low-abundance versus a high-abundance transcript.
5. Can the size of the longest clone be determined without first isolating it?
If multiple positive clones are detected, one may wish to determine their insert sizes before proceeding to their isolation. In the example shown in Fig. 4, PCR analysis using a gene-specific primer-pair (panel A) revealed positive clones in pools 1, 4 and 9 (40,000 clones/pool). Since the inserts were directionally cloned into the library vector, simultaneous analysis using the 5' vector primer and the 3' gene-specific primer (panel B) allowed determination of the extent of upstream sequences (5.5 kb in two of the clones and 3.5 kb in the other). Similarly, use of the 5' gene-specific primer with the 3' vector primer (panel C) allowed determination of the extent of downstream sequences (1.5 kb in all three clones). In this example, one could determine the insert sizes to be 6.5 kb in two clones and 4.5 kb in the other, even before proceeding to their isolation. The 6.5-kb clones turned out to be full-length.
6. What factors are important when GÇ£longGÇ¥ PCR is used with Rapid-ScreenGäó?
When the 5' vector primer is used in conjunction with a 3' gene specific primer to identify long clones, the choice of the appropriate Taq DNA polymerase is important. Fig. 5 illustrates the use of three different enzymes (labeled 1, 2 and 3), with either a 3-min (top) or a 7-min (bottom) extension time, on four selected Master Plate wells (5,000 clones/well), each containing a single positive clone with insert sizes ranging from 2.5 to 8.0 kb in length. Enzyme 1 was able to amplify all of the fragments using only a 3-min extension time, so the 7-min extension time experiment was not necessary. Enzyme 2 was not able to amplify any of the fragments with a 3-min extension time, but it could successfully amplify the 2.5- and 3.0-kb fragments when a 7-min extension time was used. Enzyme 3 partially amplified two of the fragments when a 3-min extension time was used, but it could successfully amplify all four fragments with a 7-min extension time. These experiments demonstrate the importance of choosing an efficient Taq DNA polymerase. If only small fragments are being amplified, then any of the three enzymes tested are suitable. To amplify longer fragments, a more efficient Taq DNA polymerase and/or a longer extension time is required.
7. Can Rapid-ScreenGäó Panels be used to clone different forms of an alternatively spliced transcript?
Rapid-ScreenGäó panels can be used to clone alternatively spliced transcripts, even if the desired spliced variant is many times less abundant than its parent transcript. The detection of different sized fragments by using the 5' vector primer plus the 3' gene specific primer is sometimes attributable to alternative-splicing rather than to truncated transcripts (if the alternate splicing has already been characterized, then gene-specific primers that differentiate between the two spliced forms can be designed). As shown in Fig. 6, a pair of gene-specific primers was used with the pooling technique to detect two alternatively spliced transcripts of a particular gene. The shorter form was much more abundant than the longer one; the former appeared in seven of the twelve column pools (40,000 clones/pool) and four of the eight row pools (60,000 clones/pool). The longer form, which is about 210 bp longer, was found only in the column 1 pool and in the row H pool. Thus well H1 of the Master Plate contains the lower abundant transcript variant.