cDNA cloning plays a major role in current molecular biology.1-7 Construction of a cDNA library is a highly sophisticated technology that involves a series of enzymatic reactions. The quality and integrity of a cDNA library greatly influence the success or failure in isolation of the cDNAs of interest.2 General principles begin with an mRNA transcribed into the first-strand DNA, called a complementary DNA or cDNA, which is based on nucleotide bases complementary to the mRNA template. This step is catalyzed by AMV reverse transcriptase using oligo(dT) primers. The second-strand DNA is copied from the first-strand cDNA using DNA polymerase I, thus producing a double-stranded cDNA molecule. Subsequently, the double-stranded cDNA is ligated to an adapter and then to an appropriate vector via T4 DNA ligase. The recombinant vector-cDNA molecules are then packaged in vitro and cloned in a specific host, generating a cDNA library. Specific cDNA clones can be "fished" out by screening the library with a specific probe.14-10

Traditionally, cDNA cloning takes advantage of the 3' hairpins produced by AMV reverse transcriptase during the synthesis of the first-strand cDNAs. The hairpins are utilized to prime the second-strand cDNA that is catalyzed by Klenow

DNA polymerase and reverse transcriptase. Hence, SI nuclease is needed to cleave the hairpin loops. However, the digestion is difficult and causes a low cloning efficiency and the loss of significant sequence information corresponding to the 5' end of the mRNA.2 To solve this problem, 4 mM sodium pyrophosphate is utilized to suppress the formation of hairpins during the synthesis of the first-stranded cDNA. The second-strand cDNA is then produced by RNase H to create nicks and gaps in the hybridized mRNA template, generating 3' OH priming sites for DNA synthesis using DNA polymerase I. Following treatment with T4 DNA polymerase to remove any remaining 3' protruding ends, the blunt-ended, double-stranded cDNAs are ready for ligation with adaptors or linkers. Therefore, SI digestion is eliminated and the cloning efficiency is much higher than that obtained with the classic method. Besides, the sequence information is optimally reserved.

The general strategies for cDNA cloning can be grouped into two classes. One is random cloning; the other is orientation-specific cloning.1 The random or classical cDNA cloning uses oligo(dT) as a primer and 1gt10 as cloning vectors (Figure 3.1A). The cDNA is cloned at the unique EcoR I site. Because of the single EcoR I site for cloning, the cDNAs are cloned in a random manner in sense and in antisense orientations. The constructed cDNA library can be screened using a DNA or RNA probe but not antibodies. In random cDNA cloning, if the expression vector Igtll (Figure 3.1B) is used for a cDNA library, the library can be screened using a specific antibody as well as a DNA or RNA probe. The disadvantage of using antibodies is that the possibility of obtaining the positive clones is at least 50% less than it actually is. This is because approximately 50% of cloned cDNAs are expressed as antisense RNA that can interfere with sense RNA by inhibiting the translation of the sense RNA to proteins. This shortcoming is eliminated in the orientation-specific or directional cDNA cloning using a primer-adapter to prime the synthesis of the first-strand cDNA and the use of Igtll as cloning vectors (see Construction and Screening of a Complete Expression cDNA library in Part B).

The primer-adapter consists of oligo(dT) adjacent to a unique restriction site (Xba I or Not I). The double-stranded cDNAs with EcoR I adaptors at the ends are digested with Xba I or Not I. As a result, the digested cDNAs contain two incompatible termini: one EcoR I and one Xba I or Not I. In this way, self-ligation of adapter-cDNA molecules is greatly eliminated. These molecules are readily ligated with vectors containing the same restriction enzyme termini (see Figure 3.6 for details). Compared with the random cloning method, directional cDNA cloning is much more powerful and valuable. A factor of two is the likelihood of expressing the cDNA inserts as the correct polypeptides using Igtll Sfi-Not I. In theory, no antisense RNA interference will occur in the directional cDNA cloning approach.

More advanced lambda vectors are available, in which promoters of T7, T3 or SP6 RNA polymerase are included adjacent to the multiple cloning sites (Figure 3.1C). All cDNAs can be cloned in the same direction downstream from T7, T3 or SP6 promoter. Sense or antisense RNA probes can be produced in vitro, and can be utilized as RNA probes for screening a cDNA or genomic DNA library or for northern blot analysis.

Based on the author's experience, three steps are critical for the success or failure in constructing a cDNA library. The first is the purity and integrity of the mRNAs

Hind III i_

EcoR I

Sma I

Cloning site

EcoR I

Not I EcoR I BamH I

lac Z

lac Z

FIGURE 3.1 Structural maps of vectors used for cDNA cloning.

used for the synthesis of the first-strand cDNAs. Any degradation or absence of specific mRNAs will result in partial-length cDNAs or complete loss of the specific cDNAs, especially for some rare mRNAs. The second important step is to obtain full-length cDNAs. If this procedure is not performed well, even with a very good mRNA source, the cDNA library is not so good. In that case, only partial-length cDNAs or no positive clones at all may be "fished" out. Once double-strand cDNAs are obtained, they are much more stable compared with mRNAs. A third essential step in cDNA cloning is the ligation of cDNAs with adaptors to vectors. If the ligation fails or is of low efficiency, in vitro packaging of recombinant iDNAs cannot be carried out effectively. As a result, the plaque-forming units' (pfu) number or efficiency will be very low. In that case, a poor cDNA library is not recommended for screening specific cDNA clones. In order to construct an excellent cDNA library, the following precautions should be taken. Elimination of RNase contamination must be carried out whenever possible. Procedures for creating an RNase-free laboratory environment for mRNA and cDNA synthesis include:

1. Disposable plastic test tubes, micropipet tips and microcentrifuge tubes should be sterilized.

2. Gloves should be worn at all times and changed often to avoid finger-derived RNase contamination.

3. All glassware and electrophoresis apparatus used for cDNA cloning should be separated from other labware.

4. Glassware should be treated with 0.1% diethyl pyrocarbonate (DEPC) solution, autoclaved to remove the DEPC and baked overnight at 250°C before being used.

5. Solutions to be used with mRNA and cDNA synthesis should be treated with 0.1% DEPC (v/v) to inhibit RNase by acylation.

6. It is recommended to add DEPC to solutions with vigorous stirring for 20 min. The solutions should be autoclaved or heated at 70°C for 1 h to remove the DEPC. It should be noted that because DEPC reacts with amines and sulfhydryl groups, reagents such as Tris and DTT cannot be treated directly with DEPC. These solutions can be made using DEPC-treated and sterile water. If solutions such as DTT cannot be autoclaved, the solution should be sterile-filtered.

Construction of a cDNA library is a highly sophisticated procedure. One should have a strong molecular biology background and intensive laboratory experience in molecular biology when working on this type of library. In spite of the fact that some commercial kits or cDNA libraries are available, often, many laboratories need to construct a specific library with the mRNAs expressed in a specific cell or tissue type in the spatial and temporal manner. The present chapter provides detailed and successful protocols for constructing and screening a cDNA library. Two major strategies are described. One is the subtracted cDNA library in which cDNAs derived from mRNAs are expressed in a specific cell or tissue type but not in another type. The cell or tissue type-specific cDNA clones are greatly enriched in the library, which allows specific cDNAs copied from rare mRNAs to be readily isolated. The other is the complete expression cDNA library that includes all cDNA clones from all mRNAs in a specific cell or tissue type. The step-by-step protocols described in this chapter allow experienced workers as well as beginners to construct their own cDNA libraries.

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