Many of the experiments described throughout this book require that the DNA sequence corresponding to a gene has been isolated, so it can be manipulated in vitro. The various processes involved in these in vitro experiments are collectively referred to as molecular cloning. The goal of molecular cloning is to obtain enough identical copies of a DNA sequence to allow its analysis.
A gene is considered to be cloned when its corresponding DNA sequence is readily available as a reagent, much like any other chemical or molecular reagent that can be used for experiments. Because it was rarely possible or useful to have a single molecule of the cloned gene as the reagent, the DNA sequence was often synthesized in vitro to produce many copies. Some of the enzymes described in this chapter have proved to be important tools for molecular cloning and in vitro DNA synthesis. Several of these tools are still in widespread use, while others have been largely supplanted by polymerase chain reaction (PCR), described in Tool Box 4.1. In this box, we provide a brief description of some other uses for these enzymes associated with replication.
DNA ligase to connect molecules
DNA ligase is among the most versatile enzymatic tools in molecular biology. There are many experiments in which two different DNA molecules need to be covalently connected; for example, when a cloned fragment of DNA is inserted into a plasmid, the sites of insertion have small gaps that need to be sealed. This is the role of DNA ligase—to make covalent bonds between the backbones of DNA molecules to “tie” them together. Most molecular biology labs have tubes of DNA ligase in the freezer that can be used whenever two different two molecules need to be covalently connected to each other.
Topoisomerase and DNA cloning
The biological role of topoisomerases is to unwind supercoiled double- stranded DNA molecules. In order to do this unwinding, topoisomerases cut the DNA, pass the molecule through the cut or nick, and then reseal the cut or nicked DNA. It is this cutting and resealing activity (rather than the unwinding activity) that provides the basis for a technique known as TOPO cloning; the molecular biology company Invitrogen markets a widely used TOPO-TA cloning kit based on topoisomerase. TOPO cloning, in general, uses the resealing activity of topoisomerase in a similar way to ligase. TOPO-TA cloning takes advantage of the fact that Taq polymerase used in PCR adds an extra A to the 3′ of its products; thus, if another DNA molecule ends with an overhanging T, the T from one product can base pair with the A from the other product, but the two products are not joined together, except by the T–A base pair. Topoisomerase is then added to seal the ends and connect the two products. TOPO cloning is one of the most foolproof and fast methods to clone and connect an amplified PCR product into a plasmid vector. TA cloning can also be accomplished using DNA ligase and a vector with A overhangs.
Reverse transcriptase is used to make cDNA
The protein component TERT of telomerase belongs to a class of enzyme known as a reverse transcriptase (RT). Reverse transcriptases are a type of polymerase that can use an RNA template to make a DNA copy; this is known as an RNA-dependent DNA polymerase activity. Most reverse transcriptases are encoded in the genome of viruses that have an RNA genome such as Moloney murine leukemia virus (M-MLV) or the avian myeloblastosis virus (AMV); the reverse transcriptase is used upon infection to make a DNA copy of the RNA genome, which can integrate into the DNA genome of the host.
As a research tool, RT is used to make DNA copies of mRNA and other RNA molecules in the cell. RNA degrades very easily during routine handling in the lab, whereas DNA is extremely stable. Thus, investigators will often isolate the mRNA from a cell or tissue and immediately use RT to make a single-stranded DNA copy of it. This DNA copy of the RNA, made in vitro using RT, is referred to as complementary DNA or cDNA. (We discuss cDNA in more detail in Tool Box 2.4.) Since the single-stranded cDNA copied from the mRNA would have the sequence of the non-coding or template strand, rather than the coding strand, the isolation of cDNA usually includes a second round of replication, this time using the single- stranded cDNA to make the second strand and thus the more stable double-stranded cDNA. Most RTs also include the DNA-dependent DNA polymerase activity that makes the second strand.
Using RTs to make cDNA is standard practice in most molecular biology labs. For a eukaryotic gene, it is important to note that the cDNA copy will be a copy of the mRNA, so it will include the proteincoding region, as well as the ends, but will not include the introns.