If we want to clone a cDNA from just one mRNA whose sequence we know, we can use a type of PCR called reverse transcriptasePCR (RT-PCR). The main difference between this procedure and the PCR method is that we are starting with an mRNA instead of a double-stranded DNA. Therefore, we need to start by converting the mRNA to DNA.
As usual, we can do this with reverse-transcriptase: We reverse transcribe the mRNA to make a single-stranded DNA to double-stranded. Once we have reached this stage, we use standard PCR to amplify the cDNA we have made until we have enough to clone.
We can even add restriction sites to the ends of the cDNA by using primers that contain these sites. If we use two restriction enzymes, the PCR product is a cDNA with two restriction sites at its two ends. Then we cut the PCR product with the two restriction enzymes, creating sticky ends that we can ligate into the vector of our choice. Having two different sticky ends allows us to do forced cloning, so the cDNA will have one of two possible orientations in the vector. This is very useful when we clone a cDNA into an expression vector, because the cDNA must be in the same orientation as the promoter that drives transcription of the cDNA. However, there is a caveat: We must make sure that the cDNA itself has neither of the restriction sites we added to its ends. If it does, the restriction enzymes will cut within the cDNA, as well as at the ends, and the products we get will be useless.
What kind of vector should we use to ligate to our cDNA or cDNAs? Several shoices are available, depending on the way we wish to detect positive clones (those that bear the cDNA we want). We can use a plasmid or phagemid vector such as pUC or pBS; if we do, we usually identify positive clones by colony hybridization with a radioactive DNA probe. This procedure is analogous to the plaque hybridization.
Or we can use a λ phage, such as λgt11, as a vector. This vector places the cloned cDNA under the control of a lac promoter, so that transcription and translation of the cloned gene can occur. We can then use an antibody to screen directly for the protein product of the correct gene. Alternatively, we can use a polynucleotide probe to hybridize to the recombinant phage DNA.
