Cloning DNA

We want to prepare Subcloned DNA templates, i.e. grow multiple copies of a given piece of DNA for further subcloning and/or sequencing. The following properties are desirable in a subcloning procedure, in the order of importance. Note that the second property is relevant only when the next step is sequencing rather that further subcloning.

1. To be able to represent all regions of genome (i.e. with minimal or no cloning bias)
2. Produce sequence ready DNA templates (i.e. we don't want to have difficulties with preparing the template for sequencing)

As a results we want to end up with a Genomic Library : a collection of DNA clones that covers the entire genome. We should also be able to order clones along the genome, i.e. to determine the relative positions of the clones (Physical mapping).

Idea: Recombinant DNA technology
Suppose we are presented with a given genome and we are after its base composition. Its DNA is also referred to as a foreign or donor DNA.

The idea is that we are going to create recombinant DNA by cutting the donor DNA, inserting a given fragment into a small replicating molecule ( vector) which, under certain conditions, will then amplify the fragment along with itself, resulting in a molecular clone of the inserted DNA molecule.The vector molecules with their inserts are called recombinant DNA because they represent combinations of DNA from the donor genome with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA structure is then used to transform bacterial cells and it's common for a single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are then plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells all carrying the same recombinant vector. Therefore an individual colony represents a very large population of identical DNA inserts and this population is called a DNA clone.

Procedures:

1. Isolating DNA
   The first step is to isolate donor and vector DNA. Methods for isolating genomic DNA (and this is the type which we need to obtain from the donor) had been around long before invention of recombinant DNA technology. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors and these must be purified away from the bacterial genomic DNA. One of the possible protocols is based on the observation that that at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA but the plasmids stay in the solution.
2. Cutting DNA
   The discovery and characterization of restriction enzymes made the recombinant DNA technology possible. Restriction enzymes are produced by bacteria as defense mechanism against phages. In other words they represent bacteria immune system. The enzymes inactivate the phage by cutting up its DNA at the restriction sites. Restriction sites are specific target sequences which are palindromic (both strands have the same nucleotide sequence but in antiparallel directions)) and this is one of many features that makes them suitable for DNA manipulation. Any DNA molecule will contain the restriction enzyme target just by chance and therefore may be cut into defined fragments of size suitable for cloning.

   For example the restriction enzyme EcoRY (from E. coli) recognizes the following six-nucleotide-pair sequence in the DNA of any organism:

   5'--GAATTC--2'
   3'--CTTAAG--5'
   

   The enzyme cuts within this sequence in pair of staggered cuts between the G and the A nucleotides:

   5'--G|AATT C--3'         5'--G          AATTC--3'
        |           ---->>     
        |____          
   3'--C TTAA|G--5'         3'--CTTAA          G--5'
   

   The staggered cut leaves a pair of identical single--stranded ``sticky ends''. The ends are called sticky because they can hydrogen--bond (stick) to a complementary sequence. Now, if two different DNA molecules (say donor and vector DNA fragments) are cut with the same restriction enzyme, both will produce fragments with the same complementary sticky ends making it possible for DNA recombinants to form.

   Some enzymes make staggered cuts like EcoRI whereas others make flush cuts. Flush cuts can be altered to make them suitable for making recombinant DNA.

   DNA also can be cut by other methods such as, for instance, mechanical or hydrodynamic shearing or sonication. Different methods are used at different stages of subcloning at different laboratories.

3. Joining DNA
   Donor DNA and vector DNA are digested with the same restriction enzyme and mixed in a test tube in order to allow the ends to join to each other and form recombinant DNA. At this stage the sugar-phosphate backbones are still not complete at two positions at each junction. However, the fragments can be linked permanently by the addition of the enzyme DNA ligase, which creates phosphodiester bonds at the joined ends to make a continuous DNA molecule. One of the problems of free availability of sticky ends in solution is that the cut ends of a molecule can rejoin rather than form recombinant DNA. In order to combat the problem, the enzyme terminal transferase is added. It catalyzes the addition of nucleotide ``tails'' to the 3' ends of DNA chain. Thus, ddA (dideoxiadenine) molecules are added to, say vector DNA fragments and dT molecules are added to donor DNA fragments, only chimeras can form. Any single stranded gaps created by restriction cleavage are filled by DNA polymerase 1 and the joins subsequently sealed by DNA ligase.
4. Amplifying Recombinant DNA
   Recombinant plasmid DNA is introduced into host cells by transformation. In the host cell, the vector will replicate in the normal way, but now the donor DNA is automatically replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles of cell-division will occur and the recombinant vector will undergo more rounds of replication. The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified copies of a single donor DNA is the DNA clone. (A.J.F. Griffiths et.al,[1])