It is not possible to clone mRNA directly, so it has to be converted into DNA before being inserted into a suitable vector. This is achieved using the enzyme reverse transcriptase (RTase; see Section 4.2.2) to produce complementary DNA (also known as copy DNA or cDNA). The first step in cloning from mRNA is to convert the mRNA into double-stranded complementary DNA (cDNA; also known as copy DNA) using the enzymes reverse transcriptase and DNA polymerase. The classic early method of cDNA synthesis utilises the poly(A) tract at the 3’ end of the mRNA to bind an oligo(dT) primer, which provides the 3’-OH group required by RTase (Fig. 6.2). Given the four dNTPs and suitable conditions, RTase will synthesise a copy of the mRNA to produce a cDNA • mRNA hybrid. The mRNA can be removed by alkaline hydrolysis and the single-stranded (ss) cDNA converted into double-stranded (ds) cDNA by using a DNA polymerase. In this second-strand synthesis the priming 3’-OH is generated by short hairpin loop regions that form at the end of the ss cDNA. After second-strand synthesis, the ds cDNA can be trimmed by S1 nuclease to give a flush-ended molecule, which can then be cloned in a suitable vector.
Several problems are often encountered in synthesising cDNA using the method just outlined. First, synthesis of full-length cDNAs may be inefficient, particularly if the mRNA is relatively long. This is
a serious problem if expression of the cDNA is required, as it may not contain the entire coding sequence of the gene. Such inefficient full-length cDNA synthesis also means that the 3’ regions of the mRNA tend to be over-represented in the cDNA population. Second, problems can arise from the use of S1 nuclease, which may remove some important 5’ sequences when it is used to trim the ds cDNA.
More recent methods for cDNA synthesis overcome the afore-mentioned problems to a great extent, and the original method is now rarely used. One of the simplest adaptations involves the use of oligo(dC) tailing to permit oligo(dG)-primed second-strand cDNA synthesis (Fig. 6.3). The dC tails are added to the 3’ termini of the cDNA using the enzyme terminal transferase. This functions most efficiently on accessible 3’ termini, and the tailing reaction therefore favours full-length cDNAs in which the 3’ terminus is not ‘hidden’ by the mRNA template. The method also obviates the need for S1 nuclease treatment and, thus, full length cDNA production is enhanced further.
As we have already mentioned in a number of contexts, many sup-pliers now produce kits for cDNA synthesis and cloning. Often these have been optimised for a particular application, and the number of steps involved is usually reduced to a minimum. In many ways the mystique that surrounded cDNA synthesis in the early days has now gone, and the techniques available make full-length cDNA synthesis a relatively straightforward business. The key to success is to obtain good-quality mRNA preparations and to take great care in handling these. In particular, contamination with nucleases must be avoided.
Although the poly(A) tract of eukaryotic mRNAs is often used for priming cDNA synthesis, there may be cases where this is not appropriate. Where the mRNA is not polyadenylated, random oligonucleotide primers may be used to initiate cDNA synthesis. Or, if all or part of the amino acid sequence of the desired protein is known, a specific oligonucleotide primer can be synthesised and used to initiate cDNA synthesis. This can be of great benefit in that specific mRNAs may be copied into cDNA, which simplifies the screening procedure when the clones are obtained. An additional possibility with this approach is to use the PCR to amplify the desired sequence selectively.
Having generated the cDNA fragments, the cloning procedure can continue. The choice of vector system - plasmid or phage, or perhaps cosmid or phagemid - will probably have been made before beginning the procedure or will have been determined by the manufacturer of the cloning kit. Examples of cloning strategies based on the use of plasmid and phage vectors are given next.
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Tiêu đề: Chủ đề tiếp theo: synthesis of cDNA 
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