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An efficient cDNA amplification procedure is described for determining of the 5′ and 3′ ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5′- or 3′-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5′and 3′rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of “vectorette PCR” and “suppression PCR” technologies that significantly reduce background during amplification. The application of “long and accurate PCR” (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5′- and 3′-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3–1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the “target” DNA. In the second stage, a mouse liver cDNA library with unique linker–adapter ends, referred to as the “source” cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.

The specificity and DNA yield of PCRs are often improved by the "hot start" technique and analogous methods. The intent of the approach is to eliminate or prevent the generation of nonspecific PCR templates that may be synthesized at ambient temperature prior to thermal cycling. Monoclonal antibodies (MAbs) raised in mice to the purified DNA polymerase of Thermus aquaticus (Taq) were selected for their ability to reversibly block polymerase activity. The MAbs, incubated with Taq DNA polymerase and added to PCR tubes at ambient temperature, yield specific DNA fragments upon amplification when using high numbers of temperature cycles and a very low copy number of target DNA in a complex DNA background. This approach, using the TaqStart Antibody, permits the preparation of reaction mixtures at ambient temperatures without the subsequent opening of reaction tubes, use of grease or waxes, or of degradative enzymes and deoxyribonucleotide analogs.

A rapid and reliable method is described for preparing competitive DNA fragments for quantitative PCR. Synthetic DNAs complementary to previously established PCR primers are ligated together with the primers to both ends of a generic DNA fragment whose length differs from the natural target gene PCR product. After a short ligation step, the properly constructed ligation products (i.e., those that have the correct primer templates on opposite sides of the generic DNA fragment) are preferentially amplified by PCR. The generation of competitive PCR fragments, MIMICS, can be completed in a single day. To perform quantitative PCR, known quantities of PCR MIMICS are spiked into PCR amplification reactions containing the experimental cDNA samples. A visual or radioactive comparison of the PCR products can then be used to determine the initial quantity of target gene. We show that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels.

Human glycophorin A belongs to a family of structurally related cell surface glycoproteins which are expressed in erythroid cells. We have recently isolated several human glycophorin A-specific cDNA clones and have determined their nucleotide sequence (Siebert, P. D., and Fukuda, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1665-1669). In this report by using cDNA and exact sequence synthetic oligodeoxyribonucleotides as hybridization probes, we provide evidence that human glycophorin A and B are encoded by separate and distinct genes present as single copies in the human genome. Furthermore, we show that the expression of the glycophorin A and B genes are coordinately regulated by tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate.