Don't Clone Alone

Date: September 15, 1997 cDNA Library Kit Table and Species Chart Pre-made cDNA libraries and kits abound in the market to help you probe the secrets of gene regulation while minimizing the drudgery of cDNA library construction. The ability to analyze a cell's genetic read-out-to determine which of the 100,000 possible genes are actually being expressed in a cell or tissue-is to know what makes a cell what it is. This is a central issue in molecular biology. For decades, cDNA libraries-colle

Sep 15, 1997
Laura Defrancesco

Date: September 15, 1997 cDNA Library Kit Table and Species Chart

Pre-made cDNA libraries and kits abound in the market to help you probe the secrets of gene regulation while minimizing the drudgery of cDNA library construction.

The ability to analyze a cell's genetic read-out-to determine which of the 100,000 possible genes are actually being expressed in a cell or tissue-is to know what makes a cell what it is. This is a central issue in molecular biology. For decades, cDNA libraries-collections of DNA copies of mRNA species-have been hotly pursued as a way to get at this and questions of gene regulation. How important is this? So important that a technique for identifying rare messages was the subject of a $20 million lawsuit a few years ago (K. Young Kreeger, The Scientist, Oct 2, 1995, page 1). So important that companies have been amassing literally hundreds of cDNA libraries from species as exotic as Fugu fish and tissues as unusual as tonsils.

It is no wonder that an abundance of cDNA libraries and cDNA synthesis kits is available. The preparation of cDNA libraries is one of the trickiest procedures in all of biotechnology. Before you even start making the library, mRNA must be prepared-a task fraught with difficulties because of the ubiquity of ribonucleases. Next is a complex series of enzymatic reactions to convert RNA first to an RNA/DNA hybrid before the second DNA strand is laid down and the RNA moieties removed. Following that, the ends of the cDNAs must be made compatible with an appropriate vector through the addition of linkers or adaptors, and finally, the cDNAs are ligated to the vector, making them ready for insertion into a host.

The basic approach to preparing cDNA libraries was described in the early 1980s by two pairs of scientists whose names are inextricably linked by their pioneering work in this field-Okayama and Berg, and Gubler and Hoffman. Their approach was to selectively copy messenger RNA over ribosomal RNA, by far the more abundant cellular RNA species, by exploiting the fact that only messages have polyA tails. Using oligo(dT) as a primer for reverse transcriptase, mRNAs are copied into DNA, resulting in the formation of an RNA/DNA hybrid. Following synthesis of the first strand of DNA, a second strand is laid down with a combination of RNAse H, to remove the RNA moieties, and DNA polymerase I, to synthesize the second strand of DNA. This basic procedure has withstood the test of time. A perusal of the protocols currently in use reveals that this original methodology remains, to this day, the basis for many libraries and kits.

A number of refinements to the basic methodology, however, have been made, addressing several common trouble spots:

Full-length cDNAs: For various reasons, not the least of which is the quality of the starting RNA, obtaining full-length cDNAs was a rarity. A number of companies now offer kits for mRNA isolation, which provide convenient and consistent methods for preparing high quality RNA. In addition, changes to basic cDNA technology have resulted in longer cDNAs. By incorporating long distance PCR (LD PCR) into its kits and cDNA library construction, CLONTECH reports cDNAs up to 9 kb in length. Lock-docking oligo(dT) primers eliminate 3' heterogeneity by anchoring the primer to the message end of the dT tail, thereby increasing the likelihood of generating full-length copies.

Directional insertion: Orienting the cDNA product is important to those intending to do expression screening, a process whereby the presence of a particular message in a library is determined by looking for a protein product in an expression system. Using an oligo(dT) primer with asymmetric ends, the number of clones that needs to be screened is reduced by a factor of two.

Time: Given the complexity of the technique, generating a cDNA library can easily take weeks. Pharmacia offers the TimeSaver¦ cDNA Synthesis Kit, which takes you from RNA to plating in a day. For those on a slightly slower track, Pharmacia offers the same basic kit less the time- saving devices for a two-day prep (and savings of almost $100).

Rare mRNA species: One challenge in cDNA library construction is ensuring that messages other than the most abundant mRNA species are represented in the library. The earliest experiments on cDNAs focused on messages like globin, the predominant message in blood cells. After the initial excitement wore off, researchers turned their attention to the challenge of isolating rarer messages. Today, kits and libraries using "subtractive hybridization," a method that eliminates abundant mRNA species from libraries through a set of hybridizations, are commercially available from several companies.

Amount of mRNA: Normally, 5-20 mg of mRNA is needed to generate a high quality representative library. A new technology introduced by CLONTECH enables construction of full- length cDNAs from nanograms of total RNA, using a unique "switching" oligonucleotide in the first-strand reaction.

How do you approach making a cDNA library? First, check out the pre-made libraries and save yourselves potentially hundreds of hours of work. You may be surprised to find that just the tissue/species combination you are after is available. In addition, since companies have their reputations at stake, you can expect the libraries will be of high quality. The matrix that follows will help you to determine if this is the case. The next option is to purchase a cDNA synthesis kit, of which there are now several dozen available. cDNA synthesis kits will provide you with everything you need to prepare vector-ready cDNA, short of providing starting mRNA. Lastly, custom-made libraries and arrays are offered by a number of companies listed later in this article.

From apes to zebrafish, and from nearly every tissue and stage of development, remarkable collections of cDNA libraries are commercially available. Some companies, CLONTECH and Stratagene among them, have amassed huge banks of cDNA libraries that cover the gamut of species and tissues. Other companies, like Genpak and Novagen, have focused on a few specialized areas. Arrayed cDNA clones are available from the I.M.A.G.E. Consortium, which collects and arrays human and mouse cDNA libraries (see box). The consortium has the largest public collection of partially sequenced cDNA clones, with libraries and individual clones distributed by five companies for a nominal fee.

cDNA libraries can be made in either phage vectors or plasmid vectors; as detailed in the following section, there are roughly equal numbers of each kind of library. Each vector has its advantages. Traditionally, lambda phage vectors have been chosen because they can accommodate large inserts, though this is more of an issue for genomic libraries. Phage vectors also have high cloning efficiencies and plating densities, making them preferable when looking for rare message species. In addition, a number of lambda vectors are engineered for expression in bacteria; Stratagene's ZAP Express¦, for example, allows for both bacterial and eukaryotic expression and has the additional property of allowing in vivo excision of inserts. On the other hand, plasmid vectors are easier to handle, they are more flexible for post-cloning manipulations, and many are engineered for eukaryotic expression.

Type Culture Collection offers numerous cDNA libraries constructed by scientists and contributed to the collection. They represent a range of species and tissues and are in a variety of vectors. Also offered are cDNA clones from the I.M.A.G.E. Consortium as well as human cDNA clones sequenced by The Institute for Genomic Research (TIGR). Available within the human cDNA clone set are over 1,000 full-length human cDNA clones. Individual libraries cost between $158 and $236. Clones are available for $24 to $137.

CLONTECH produces cDNA libraries according to the method of Gubler and Hoffman, using size-selected (>500 base pairs) cDNAs cloned either into plasmid or lambda vectors. Libraries cloned into Lambda TriplExTM, a phagemid that expresses cloned inserts in all three reading frames, are ideal for immunoscreening. 5'STRETCH PLUS¦ cDNA libraries are prepared using a unique oligo(dT)25d(A/C/G) primer during first-strand synthesis and a specially designed EcoR1 hemiphosphorylated adaptor for subsequent cloning. Sequence representation of all 5'STRETCH PLUS cDNA libraries is estimated by hybridization using a human beta-actin cDNA probe; all human cDNA libraries must show a minimum beta-actin frequency of 0.10%, and all other mammalian cDNA libraries must show a minimum beta-actin frequency of 0.05%.

DNA Technologies (DTI) offers several pre-made cDNA libraries. cDNA libraries are prepared from human, rat, or mouse tissues by inserting size-selected cDNAs (>400 bp) into lgt11 or lgt10 vectors. More than 90% of the clones contain inserts. DTI also offers custom cDNA library construction services. Libraries can be prepared in plasmid and/or lambda vectors of choice for either prokaryotic or eukaryotic expression studies.

Genome Systems offers cDNA clones from the I.M.A.G.E. Consortium in a number of formats including individual clones, 384 well plates, and high-density filters. The company also provides access to zebrafish, Toxoplama gondi and Drosophila cDNA clones. It is initiating a high- throughput cDNA screening service,which will provide the longest cDNA clone from in-house libraries within days. The company currently will provide access to 12-15 libraries encompassing tissues from human, rat, mouse, and Drosophila. The number of libraries offered is expected to increase throughout the next year.

Genpak Genpak's cDNA libraries focus on cancer and the immune system, with unique libraries from sources such as human natural killer T cells and small cell carcinoma. All libraries are constructed with the aim of being full length, and are cloned into the multiple cloning site of pCDM18. This vector has the SV40 replication origin and CMV viral promoter for transfection into mammalian cells for expression studies. Libraries are not amplified and are supplied as a ligation mix together with the bacteria so that the user can obtain and keep a primary library. First strand synthesis is primed by oligo(dT) with the second synthesized by the procedure of Gubler and Hoffman, giving rise to representative and full length cDNA libraries. Each library costs $550.

Invitrogen offers several pre-made libraries from human, rat, mouse, and wheat cells and tissues in pcDNA3.1, a eukaryotic expression vector. Invitrogen's pre-made cDNA libraries offer representation and ease of use. All libraries have a guaranteed 1 x 106 primary number of recombinants and are checked for inserts and average insert size. First-strand cDNAs of all libraries are tested by PCR to ensure representation of housekeeping genes (actin and clathrin) and are amplified on plates only once to reduce loss of representation. Libraries are supplied ready to plate and are accompanied by an extensive support guide. Invitrogen prices its libraries at $700.

Pre-made SuperScript directional human and mouse cDNA libraries are made by Life Technologies in the pCMVSPORT 1 vector. This vector contains a CMV promoter for expression analysis, an f1 origin of replication for single-stranded DNA production, and SP6 and T7 polymerase promoter sites for RNA synthesis. First-strand synthesis is catalyzed by SuperScript II RNAseHo reverse transcriptase with which average insert sizes of 1.3-1.9 kb are obtained, making full-length clones likely. Life Technologie's pre-made libraries are priced at $625.

Novagen offers a collection of cDNA libraries featuring different development stages and tissues of Drosophila and mouse. All cDNA libraries are constructed in Novagen's lox autosubcloning vectors by directional cloning strategies. cDNAs are size fractionated such that inserts are greater than 500 base pairs; size ranges have been verified to be from 500 base pairs to greater than 4,000 base pairs. Novagen libraries are priced at $450.

In addition to I.M.A.G.E. Consortium cDNA clones, Research Genetics offers normalized and non-normalized human and mouse cDNA libraries constructed using a patented method for normalization based, in part, on a Cot curve hybridization. Libraries are subject to certain restrictions: these cDNA libraries are sold under license from Columbia University. Rights to these libraries are limited to internal research use by academic or nonprofit institutions only. Research Genetics also markets cDNA libraries from the model organisms, zebrafish and Xenopus, with the option of purchasing them on membranes. Unarrayed libraries are priced between $1,500 and $2,740, while the membranes cost $250-$500.

Stratagene offers a collection of cDNA libraries in the Lambda ZAP" II and ZAP Express vectors, which allow for easy manipulation of single clones and mass excisions of inserts by the ExAssist¦ excision system. All bacterial strains needed to screen and characterize the libraries are included. Libraries may be primed with non-directional oligo(dT), directional oligo(dT) for expression screening, or non-directional oligo(dT) plus random primer. Most Stratagene libraries cost $730; some exceptions run over $1,000.

The table below is a compilation of all cDNA libraries currently on the market. In addition to these pre-made libraries, custom-made libraries are available from Advanced Genetic Technologies Corp., Paragon Biotech Inc, CLONTECH, PerImmune, Inc., DNA Technologies, Primm Labs, Genpak Inc., Rabbit and Rodent Diagnostics, Life Technologies Research Genetics, Molecular Cell Science, SouthWest Scientific Resources, Molecular Diagnostic Science , Stratagene, and NBL/Genovus.

This set of research tools provides the materials to perform the first steps in preparing a cDNA library from an mRNA preparation of your own making. Most kits listed provide the materials for first- and second-strand synthesis, yielding so-called "vector ready" double-stranded cDNAs. Kits differ in the final form of the cDNA: some of the cDNAs are designed for insertion into any vector, while others prepare cDNAs for use with a particular vector. Often the latter kits will come supplied with the vector. Linkers/adaptors may come in the kits, along with material to purify the cDNAs after linker/adaptor attachment, while others produce flush-ended cDNA to which any linker may be attached. Finally, some manufacturers offer two kinds of kits: those that produce the cDNAs and those that take you through library construction with vector and packaging extracts included.

With the advent of new technologies have come alternative formats for making and manipulating cDNA libraries. Some of the latest cDNA emanations are discussed below:

PCR-Ready cDNAs: PCR enables direct amplification and sequencing of cDNAs without having to clone them first. Accordingly, a new generation of research tools that either provide pre-made PCR-ready cDNAs or the materials to make them are now available. Pre-made first-strand cDNAs from unique sets of tissues are offered from CLONTECH, Invitrogen, and Maxim Biotech. Mixtures of PCR ready cDNAs from different tissues are offered by OriGene Technologies in its Multiple Choice¦ cDNAs, which is a collection of six sets of tissue-specific cDNAs. Finally, kits for the preparation of first strand cDNAs are becoming abundant on the market, with roughly a dozen currently offered.

Several unique products in this category are also in the market. CPG offers the SOLIDscript¦ Solid Phase cDNA Synthesis Kit, which conducts cDNA synthesis on immobilized streptavadin- coated magnetic particles to which your mRNA will be bound. The immobilized, single-stranded cDNA is ready for PCR, subtractive hybridization, RACE, cDNA amplification, or cloning. Pharmacia's Ready-To-Go" You-Prime First-Strand Beads combine all the components for first- strand synthesis in a single-reaction, room temperature-stable format. Following the first-strand reaction, the product is ready for PCR amplification or standard second-strand synthesis.

cDNA Arrays: Several companies offer defined arrays of cDNAs on membranes or matrices, custom designed or pre-made, ready for analysis with tissue-specific probes. Arrays can be useful for quick screening with probes of interest. In addition, in some cases, it is possible to purchase individual clones that react with your probes.

CLONTECH's AtlasTM human cDNA expression array has 588 cDNAs affixed to nylon membranes with each quadrant defined by a particular function such as oncogenes or proteins involved in cell to cell communication. Synteni will custom build arrays of cDNAs made with user-provided mRNA on a glass matrix, Gene Expression Micro Array, or GEM; Affymetrix has an expression cDNA chip that arrays probes for 6,000 to 7,000 genes for analysis in its semiconductor-based GeneChip¦ System. Genome Systems Inc. and Research Genetics prepare high-density hybridization filters and cDNA pools from human and mouse species for screening. Research Genetics has recently added GeneFilters¦, which contain PCR products from yeast ORFS and I.M.A.G.E. clones, spotted onto nylon filters for analysis of gene expression. The I.M.A.G.E. libraries, described here in this article, array individual cDNA clones in microtiter wells, and the consortium will soon release through its distributors a nonredundant collection of IMAGE cDNA clones representing unique genes. Both ATCC (EXPRESS-CHECK¦ Kit) and CLONTECH (QUICK-Screen¦ Human cDNA Library Panel) provide collections of libraries for expression analysis.

The future of the study of the regulation of gene expression may well be in cyberspace, not at the lab bench, as in silico hybridization-surfing the net for sequence homology in databases-may supplant cDNA library construction and screening as a way of finding useful cDNA clones. The Expressed Gene Anatomy Database (EGAD), part of the nonprofit The Institute for Genome Research (TIGR), for example, contains sequences for over 90,000 human expressed sequence tags. A variety of types of searches can be conducted through TIGR's website (www.tigr.org): by gene product name, by sequence, or even by cellular roles. With just a little sequence data, it's possible to locate and obtain clones of your gene of interest, getting past cloning and screening without ever going to the lab bench.

cDNA Library Kit Table and Species Chart