Over 145,000 human cDNA clones arrayed for nucleic acid screening
Jean-Michel Llias Leonardo de Leon Bruce Jerpseth
Stratagene has organized over 145,000 high-quality clones from a universal cDNA library that represent genes expressed in 29 different human tissues. This normalized library demonstrates a good average length of inserts, low representation of the ubiquitous b-actin gene, and high percentage of full-length clones. The bacterial colonies originally arrayed in 384 microwell plates have been grown individually on agar plates in pools of 96 clones. The DNAs obtained from the 1,536 pools have been double-spotted on nylon membranes the size of a microwell plate. Stratagene now provides these membranes for screening procedures as part of a powerful and unique cloning system that allows human genes to be fully isolated in a short period of time with only two hybridization steps.
The Human Universal cDNA Library (HUCL) was constructed essentially by a primer/adapter method.1 The cDNAs were synthesized independently from each mRNA source using a synthetic oligonucleotide, 5-CCCGGG(T)18-3, as a primer. The mRNAs were then removed by alkaline hydrolysis, and the remaining single-stranded cDNA molecules were (dG)-tailed. At this step, an aliquot of each sample was electrophoresed on an agarose gel for analysis, and the cDNAs corresponding to the 29 organs or tissues listed in Table 1 were selected and pooled.
Brain: Caudate Nucleus
Brain: Substantia Nigra
Brain: Subthalamic Nucleus
The total RNAs were obtained by a modified guanidium thiocyanate method,2 and the mRNAs were selected by two rounds of oligo(dT)-cellulose enrichment. Each poly(A)+ RNA has been found to display a discrete band by Northern blot analysis when hybridized with a human b-actin cDNA probe.
An aliquot of this pooled cDNA was used to construct a primary library. The cDNA was annealed with the adapter 5-(A)18CCCGGGAGCT-3, and the plasmid pT7T3 18 U that had been linearized with Pst I, (dC)-tailed and digested with Sac I. Once the single-stranded region was ligated and repaired, it produced recombinant molecules in which the inserts were bound at their 5 termini by a poly(C) and at their 3 termini by an Sac I site (reconstituted by the cohesive ends of the primer/adapter and vector). These molecules were transformed into E. coli resulting in 2 x 105 ampicillin-resistant clones.
The remaining cDNA was size-fractionated by electrophoresis in a low melting point agarose gel. One-third of the total cDNA was used to select molecules longer than 1 kb, while two-thirds was used to select for molecules longer than 2 kb. The two resulting fractions were combined to obtain a population enriched for high molecular weight cDNAs without losing representation of the smaller full-length molecules (Figure 1).
The cDNA obtained by this fractionation procedure was used in the construction of two normalized libraries.3 The corresponding mRNAs used for cDNA synthesis were pooled, biotinylated with photobiotin, and hybridized in excess with the cDNA. Streptavidin treatment, followed by phenol extraction, removed cDNA-mRNA hybrids and mRNA so that only the unhybridized cDNA remained in the aqueous phase. One-half of this unhybridized cDNA was used to construct a library of 2 x 105 clones (as previously described). The other half was used for a second round of subtractive hybridization resulting in a library of 2.5 x 104 clones (Figure 2). These two normalized libraries were directly amplified on agar plates. The individualized colonies were lifted onto membranes and duplicated onto a second set of membranes. One set was used to purify the DNA for analysis and storage while the other set was soaked in a sucrose solution and frozen at -80C to be used for further organization into microwell plates.
The frozen membranes were thawed on agar plates, and the colonies individually picked and cultured in microwell plates to be finally stored at -80C. Approximately 123,000 and 22,000 clones were arrayed from the two libraries obtained after one and two cycles of normalization, respectively. The DNA analysis of individual clones picked at random revealed an average insert length of 1.7 kb (Figure 3), which was consistent with an earlier analysis performed on over 100 clones (data not shown).
To preserve the advantage of having organized primary clones that had never been in contact with each other, the subsequent amplifications of these clones were performed by growing the colonies separately before any pooling process. The bacterial colonies were individually grown on agar plates with 96 clones per plate. The clones from each plate were then pooled to isolate the DNA, and the DNA samples obtained from the resulting 1,536 pools were double-spotted onto nylon membranes for the screening procedure (Figure 4).
The presence of the ubiquitous b-actin gene (1.8 kb)4 was analyzed in each library to evaluate the efficiency of the fractionation-normalization procedures. In this analysis, we probed the libraries with two different radiolabeled oligonucleotides.5 One probe was designed to hybridize to the beginning of the 5 translated region (ATG included) while the other corresponded to the end of the 3 untranslated region (Figure 5 and Figure 6). The percentage of full-length cDNAs was determined by comparing the two results.
In situ hybridization of 6,000 colonies from the library obtained before fractionation established that b-actin was originally represented at approximately 1 in 200 clones with 45% being full-length (data not shown). Dot blot analysis on the Master Membranes (Figure 6) showed that after fractionation and one round of normalization, the representation of this gene fell to 1 in 5,000 clones with 70% being full length. After the second round of normalization no b-actin clone was detected in the organized colonies, suggesting that, after extensive hybridization, each of the 22,000 clones arrayed could be unique.
A set of four Master Membranes, each the size of a microwell plate, allows the screening of 1,536 pools of 96 clones in the same conditions as ordinarily used for in situ hybridization on colonies. The hybridization signals are detected by autoradiography or phosphorimaging, and the positive pools are easily located by rows and columns printed on the membranes (Figure 6). When the position of a positive pool has been identified, the corresponding Secondary Membrane gridded with the individual colonies can be obtained from Stratagene for a secondary screening. The result of thesecondary screening determines the exact location of the target cDNA clone, which can then be obtained as a bacterial stab culture.
Stratagenes Human Universal cDNA Library Array I represents a collection of over 145,000 primary clones individually organized in microwell plates and stored at -80C. The high quality of these clones obtained after extensive normalization has been demonstrated by an average insert length of 1.7 kb, a representation of the ubiquitous gene b-actin 25- to 100-fold lower than in the direct library, and a high representation of full-length clones (up to 70% for b-actin). Using this material, Stratagene has developed a powerful tool to accelerate gene discovery by nucleic acid screening: A first set of four Master Membranes allows a rapid screening of pools and can be reused for different cloning projects, while a Secondary Membrane is used to identify the final position of the target cDNA clone. Human Universal cDNA Library Array I allows researchers to efficiently identify and isolate human genes by screening a large representation of high-quality cDNA clones in a short period of time.
HUCL Array I Master Membranes were hybridized using a conventional method. Prehybridization and hybridization were performed in 6X SSC buffer, 5X Denhardts reagent, and 0.1% SDS. The oligonucleotide probes were prepared using T4 polynucleotide kinase and high-specific-activity [g-32P]ATP (3000 Ci/mmol). The specific activity for the probes were approximately 5 10 8 cpm/mg, and their concentrations in the hybridization solution were 5 105 cpm/ml.
The two sets of Master Membranes were hybridized overnight at 42C, and then washed in a 6X SSC buffer and 0.1% (w/v) SDS wash solution up to 1C below the calculated Tm. The membranes were mounted with Glogos II autorad markers and exposed to x-ray film with intensifying screens at 80C for 16 hours. After exposure, the autoradiograph films were realigned on the mounted membranes to locate the positive signals.
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