New Cloning Matrix^™ System For Stratagene’s Human Universal CDNA Library

Find rare human cDNA clones with the speed of PCR and the fidelity of direct colony screening

New Cloning Matrix^™ System for Stratagene’s Human Universal cDNA Library

Mark Dickey • Alexey Novoradovsky • Adrienne Evans • Matthew Klimjack • Jean-Michel Lélias

Stratagene

The Cloning Matrix^™ system for Stratagene’s Human Universal cDNA Library (HUCL) §§ represents genes expressed in 29 different human tissues that are part of a unique collection of approximately 300,000 primary clones individually organized in microwell plates. These high-quality cDNA clones, obtained after extensive normalization, comprise an average insert size of 1.7 kb, a low redundancy of ubiquitous genes, and a percentage of full-length genes estimated to be over 50%. The clones were grown individually prior to plasmid preparation and the DNA samples were then pooled together so that the clone of interest can be quickly identified using a PCR screening strategy. This new PCR format, known as the Cloning Matrix  system, complements our powerful HUCL arrays on membranes and is the fastest way to identify and isolate cDNA clones for gene discovery when nucleic acid sequence information is available.

An enormous amount of sequence information generated from the Human Genome Project has been made publicly available, and this data is being widely used by the research community in the final race to isolate and further characterize novel human genes. Based on this information, oligonucleotides can be synthesized for screening by in situ hybridization or for direct PCR amplification from different RNA sources (RT-PCR).

With the exception of our unique HUCL arrays on membranes,¹ colony screening by hybridization is a long and tedious process involving large-scale plating of bacteria on filters with absolutely no guarantee of the end results. Although RT-PCR is a much faster process, it often results in mutation, deletion, or insertion in the cDNA and also necessitates several cloning steps to get to the physical clone. The HUCL arrays on membranes have been used successfully to rapidly isolate full-length genes that correspond to rare and/or long transcripts.^2,3 This unique collection of primary clones is now available as the Cloning Matrix system— a format that allows rapid PCR screening while maintaining the fidelity of direct colony screening. The final clone obtained by this process is not a PCR product but a cDNA clone that has been individually picked and cultured in a microwell plate when the library was first constructed.

Library Construction

HUCL was constructed by a primer/adapter method that anneals the single-stranded cDNA molecule to the vector prior to the ligation and second-strand synthesis steps.^1,4 This technique is very efficient for cloning full-length molecules  and virtually eliminates the risk of introducing artifacts commonly seen in other cDNA libraries, like cloning genomic fragments or obtaining two cDNAs fused together in the same vector.

Table 1
mRNA Sources Used to Synthesize the Single Strand cDNAs

The single-stranded cDNAs corresponding to the 29 tissues listed in Table 1 were synthesized independently from each mRNA source, then pooled together for further normalization steps.^1,5 An aliquot of this cDNA was used to construct a direct library N₀ (unnormalized clones), while the remaining material was used to construct the normalized libraries N₁ and N₂. Library N₁ comprises clones obtained after one cycle of normalization while library N₂ represents clones obtained after a second cycle.

Colonies obtained after transformation and plating on agar were not amplified as a pool but were individually picked and cultured in microwell plates. To maintain the advantage of having organized authentic primary clones, all subsequent amplification steps were always performed by growing the colonies separately to avoid competitive selection. We analyzed a large number of known sequences and noted that over 50% of these clones contained a full-length open reading frame, which elevates HUCL well beyond other libraries commonly used for gene discovery.

Screening Procedure

Fig.1

The HUCL clones have been arrayed in 768 microwell plates containing 384 clones each.  To limit the number of PCR reactions need to run, we first pooled each 384 well plate in the array. To avoid running 768 separate PCR reactions to find the plate(s) containing the cDNA target, we pooled the DNA samples prepared from each microwell-plate into three virtual dimensions (column, row, and surface). With this Cloning Matrix system, only 88 DNA samples (24 columns, 32 rows, and 32 surfaces) are needed to identify the plate(s) containing the clone(s) of interest. Each positive clone confers a positive result in one row, one column, and one surface, and the intersection of these three different coordinates identifies which plate number to order (Figure 1 and Figure 4).

Figure 4
Positive Clones Identified on a Grid Representing the HUCL Plates Organization

Each number inside the grid represents a 384-well plate stored at -80°C.

Each Cloning Matrix system is provided in a 96-well format containing DNA controls and DNA pools necessary for 10 complete screening procedures. Two vector-specific primers are also included to allow amplification of the 5' or 3' end of the cDNA insert(s).  As seen in Figure 2, these primers can be used with an appropriate gene-specific primer to obtain information on the length of the cDNA and identify the longest clone or potential splice variants. Since this screening procedure uses a vector-specific primer, it may be more delicate and sometimes results in a weaker signal than when two gene-specific primers are used (Figure 3).  However, these results can provide valuable information when choosing which HUCL plate to order if more than one cDNA target is present in the library.

Fig.2

Fig.3

In the Cloning Matrix system for HUCL, most of the arrayed clones are unique within the collection, or represented in very few wells. If just one positive clone exists in the entire array, only the column-row intersection is needed to identify the plate containing the cDNA target.  If more than one clone is present, the surface dimension is then used to eliminate any redundant coordinates at the column-row intersections. In Figure 4, plates 76 and 691 contain cDNA targets giving positive results with a specific set of primers. The PCR results given by the column-row intersections show four possibilities (76, 91, 676, and 691) but the positive results obtained on surfaces S01 and S32 eliminate the false possibilities in positions 91 and 676. To determine which 384-well plate to order after the first round of PCR, we provide online Cloning Matrix^™ system software available at www.stratagene.com.

When the position of a positive result has been identified on DNA pools, the corresponding HUCL plate containing the individual colonies can be obtained from Stratagene. These clones are provided in a 384-format as bacterial cultures frozen in a solution that allows for immediate PCR amplification. In addition, the plates may undergo multiple freeze-thawing cycles without losing viability in the bacterial stock. To avoid running 384 PCR samples to locate the position of the cDNA target, our protocol suggests pooling, by rows and columns, a few microliters of the solution contained in each well. One microliter of each pool can then be used directly as template to run the 40 PCR samples (16 rows and 24 columns using the same conditions that gave the previous positive result on DNA pools (Figure 5). The positive row and column determine the coordinates of the well containing the clone of interest.

Conclusions

Stratagene’s Human Universal cDNA Library is an exceptional collection of primary clones arrayed in microwell plates. The large panel of human tissues represented, the low redundancy of ubiquitous genes, and the very high percentage of full-length molecules make this material the most appropriate source to be used today for gene discovery. This unique library that potentially contains every human gene is now available as our Cloning Matrix System—  a PCR format that allows you to immediately identify and obtain the 384-microwell plate containing the cDNA target. The final cDNA clone obtained is not a PCR product but an authentic primary clone that can be further characterized with total confidence. This screening procedure is therefore the most efficient and reliable tool to rapidly isolate novel human genes when DNA sequence information is available.

REFERENCES

1. Lélias, J.-M., et al. (1998) Strategies 11: 29-32.
2. Costello, L.C., et al. (1999) J. Biol. Chem. 274: 17499-504.
3. Mehta, A., et al. (2000) Mol. Cell Biol. 20: 1846-1854.
4. Caput, D., et al. (1986) Proc. Natl. Acad. Sci. USA 83: 1670-1674.
5. Lélias, J.-M., et al. (1993) Proc. Natl. Acad. Sci. USA  90: 1479-1483.

§§ Purchase of these products includes a limited license to use these products for research purposes. For information on commercial use licenses, please contact Stratagene's Product Manager for libraries at (800) 424-5444.