How Larger DNA Fragments Can Be Cloned?

Both λ phage vectors and the more commonly used E. coli plasmid vectors are useful for cloning DNA fragments up to ≈20 – 25 kb. However, cloning of much larger fragments is desirable for sequencing of extremely long DNAs such as the DNA in a eukaryotic chromosome. Also, because of the common occurrence of large introns in genes from higher eukaryotes, it is often necessary to clone DNA fragments greater than 25 kb in order to include an entire gene in one clone. Consequently, additional types of cloning vectors have been developed for cloning larger fragments of DNA.
One common method for cloning larger fragments makes use of elements of both plasmid and λ phage cloning. In this method, called cosmid cloning, recombinant plasmids containing inserted fragments up to 45 kb long can be efficiently introduced into E. coli cells. A cosmid vector is produced by inserting the COS sequence from λ phage DNA into a small E. coli plasmid vector about 5 kb long. Like other plasmid vectors discussed earlier, cosmid vectors contain a replication origin (ORI), an antibiotic-resistance gene (e.g., ampr), and a polylinker sequence containing numerous restriction-enzyme recognition sites.Next, the cosmid vector is cut with a restriction enzyme and then ligated to 35- to 45-kb restriction fragments of foreign DNA with complementary sticky ends. If the concentration of foreign DNA is high enough, the ligation reaction generates long DNA molecules containing multiple restriction fragments of the foreign DNA separated by the 5-kb cosmid DNA. These ligated DNA molecules, which resemble the concatomers that form during replication of λ phage in a host cell, can be packaged in vitro as described earlier.


General procedure for cloning DNA fragments in cosmid vectors. This procedure has the high efficiency associated with λ phage cloning and permits cloning of restriction fragments.
In the packaging reaction, the λ Nu1 and A proteins bind to COS sites in the ligated DNA and direct insertion of the DNA between two adjacent COS sites into empty phage heads. Packaging will occur so long as the distance between adjacent COS sites does not exceed about 50 kb (the approximate size of the λ genome). Phage tails then are attached to the filled heads, producing viral particles that contain a recombinant cosmid DNA molecule rather than the λ genome. When these virions are plated on a lawn of E. coli cells, they bind to phage receptors on the cell surface and inject the packaged DNA into the cells.
Since the injected DNA does not encode any λ proteins, no viral particles form in infected cells and no plaques develop on the plate. Rather, the injected DNA forms a large circular plasmid, composed of the cosmid vector and an inserted DNA fragment, in each host cell. This plasmid replicates and is segregated to daughter cells like other E. coli plasmids and the colonies that arise from transformed cells can be selected on antibiotic plates. The high efficiency of λ phage infection of E. coli cells makes cosmid cloning a practical method of generating plasmid clones carrying DNA fragments up to 45 kb long. Since many genes of higher eukaryotes are on the order of 30 – 40 kb in length, cosmid cloning increases the chances of obtaining DNA clones containing the entire sequences of genes.