Generate High-Quality, Directional Plasmid CDNA Libraries

Produce larger primary libraries with new plasmid library construction kits

Generate High-Quality, Directional Plasmid cDNA Libraries

Quinn Lu • Marie Callahan • Tanya Hosfield • Barry Neiditch • Alan Greener • John C. Bauer • Mike Kobrin
Stratagene

Stratagene’s pBluescript ^Ò II, pcmv-script® and pAD-GAL4-2.1 XR library construction kits offer directional cloning of cDNAs and high-efficiency transformation into Epicurian Coli ^® XL10-Gold ^ä ultracompetent cells.* Plasmid cDNA libraries constructed with XL10-Gold cells yield larger primary libraries than libraries made with other E. coli transformation hosts, thereby increasing the probability of recovering full-length clones. Libraries constructed in these plasmid systems can be screened directly for gene homology or gene expression. Each of the three library construction kits is unique, designed specifically for prokaryotic expression, eukaryotic expression or two-hybrid §§   screening in yeast.

cDNA libraries are a central method for discovering new genes. Traditionally, genes have been selected for sequence homology; more recently, genes are being discovered by gene function. Both lambda and plasmid vectors have been used extensively to create cDNA libraries for these purposes. For functional screening applications, such as yeast two-hybrid screening, complementation studies and expressed sequence tag (EST) sequencing, cDNA libraries constructed directly into plasmid vectors are convenient and easy to use. However, constructing high-quality plasmid libraries can be limited by the efficiency of the cDNA synthesis reaction and the transformation of plasmid clones. Stratagene has improved the general methods for constructing plasmid cDNA libraries and offers three plasmid-based cDNA library construction kits using the pBluescript II (prokaryotic), pCMV-Script (eukaryotic) and pAD-GAL4-2.1 (yeast two-hybrid) vectors (table 1). In each system, directional cDNA synthesis¹ is optimized using cloned MMLV reverse transcriptase to synthesize a greater yield of high-molecular-weight cDNA. Transformation of plasmid clones is significantly improved by the use of XL10-Gold ultracompetent cells,² which generate larger primary plasmid libraries with greater complexity and more full-length clones.

Directional cDNA Synthesis

The pBluescript II, pCMV-Script and pAD-GAL4-2.1 XR library construction kits feature directional cDNA synthesis** using Stratagene’s cDNA Synthesis Kit. Primary libraries containing greater than 1 x 10⁶ primary colonies are routinely achieved with 5 µg of mRNA from a variety of tissues. Directional synthesis (figure 1) doubles the probability of expressing functional protein by ensuring that cDNA is in the proper orientation for protein expression. In this method, MMLV reverse transcriptase is used to synthesize first-strand cDNA from mRNA using a hybrid oligo(dT) linker-primer that contains an Xho I restriction site near its 5¢ end. First-strand synthesis takes place in the presence of 5-methyl dCTP and nonmethylated dATP, dGTP and dTTP. Second-strand synthesis is then performed using DNA polymerase I, cloned RNase H³ and nonmethylated dNTPs. The resulting hemimethylated cDNA is blunted using Pfu DNA polymerase,⁴ ligated to EcoR I adaptors and digested with Xho I. Since any internal Xho I sites are hemimethylated and protected from digestion, only the nonmethylated Xho I site in the linker-primer is cleaved. The resulting cDNA contains an Xho I-compatible overhang on its 3¢ end and an EcoR I-compatible overhang on the 5¢ end. After size fractionation, the cDNA is ready for directional cloning into either the pBluescript II, pCMV-Script or pAD-GAL4-2.1 vector predigested with Xho I and EcoR I (XR) restriction enzymes.

Transformation

In order to construct a complex plasmid library, synthesis of cDNA and ligation to the vector must be as efficient as possible. Ultimately, the final quality of a plasmid cDNA library is determined by transformation of the ligation products into the E. coli host. The optimal E. coli host for generating large, complex cDNA libraries was determined by comparing the primary size of cDNA libraries constructed in the pCMV-Script and pAD-GAL4 XR predigested vectors and transformed into XL2-Blue,⁵ XL10-Gold or DH10B*** cells (figure 2). The average number of primary clones obtained from transformation into XL10-Gold ultracompetent cells was 4.6-fold greater than the number of primary clones obtained from XL2-Blue cells and 22-fold greater than obtained from DH10B cells. For the tranformation control, supercoiled pUC18 was used. The transformation efficiency for XL2-Blue and XL10-Gold cells was 5 x 10⁹ colony-forming unit (cfu)/µg of pUC18 DNA, and transformation efficiency for DH10B cells was 1 x 10⁹ cfu/µg of pUC18 DNA. When comparing XL2-Blue and DH10B cells, the relative library size obtained when the pCMV-Script and pAD-GAL4 XR predigested vectors were used correlates well with the relative efficiencies observed for supercoiled pUC18 DNA. However, the relative library size obtained from transformation into XL10-Gold cells was 4.8-fold higher than predicted by the transformation efficiency observed with supercoiled pUC18 DNA.

In circumstances where only limited amounts of starting material are available for constructing a cDNA library, the highest efficiencies are required. For these instances, it may be preferable to construct the library in a lambda vector, which offers 4-5 times more primary clones per microgram of RNA. Stratagene’s unique Lambda ZAP ^® vectors allow high-titer lambda libraries to be generated, and single lambda clones or entire Lambda ZAP libraries can be rapidly converted into a plasmid format.^6-10

Conclusions

Stratagene’s new plasmid cDNA library construction kits are designed for generating high-quality, directional cDNA libraries in either the pBluescript II (prokaryotic), pCMV-Script (eukaryotic) or pAD-GAL4-2.1 (yeast two-hybrid) XR vectors. When screening cDNA libraries by functional methods, such as yeast two-hybrid screening, eukaryotic expression or antibody screening, directional cloning increases the probability of finding clones. These library construction kits are provided with Stratagene’s cDNA Synthesis Kit as well as XL10-Gold ultracompetent cells for the highest transformation efficiency of ligated DNA.

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