The Use of Cosmids as Cloning Vehicles

Abstract

The use of plasmid vectors in the cloning of very large segments of DNA is hampered by the low transformation efficiency of large plasmids. Bacteriophage lambda can be used as a vector to clone efficiently larger segments of DNA. Lambda vectors can accept at most 18–21 kilobases (kb) of foreign DNA.1 More recently Collins and Hohn2 have developed a new type of plasmid vector which combines the high efficiency of transfection with packaged ‘phage’ particles and the advantage of using a plasmid vector to accept larger segments of foreign DNA. This new type of vector differs from the usual plasmid vectors in the presence of a small piece of lambda DNA, the so-called cohesive end site. Lambda DNA in the virus is linear with two single-stranded ends of twelve nucleotides in length. Because these ends are complementary, they are called the cohesive ends or cos sites. Such vectors are, therefore, designated as cosmids. Cosmid hybrids can be packaged into lambda phage heads and this allows the cloning of the DNA fragments up to 52 kb. After transduction into a lambda-sensitive bacterium the hybrid plasmids replicate as plasmids and are selected for by using antibiotic resistance markers mediated by these vectors. The efficiency by cosmid cloning can be as high as 106 clones per μg of foreign DNA. This is comparable to the efficiencies obtained with lambda vectors. An important advantage of cosmid cloning is that the system selects specifically the cloning of large DNA fragments, in contrast to transformation, which tends to favour the cloning of smaller DNA fragments.