1-4.1 Introduction:

You now know that in recombinant DNA technology, vectors are used as carrier of foreign DNA into the host organism. Apart from bacterial plasmids, several other modified vectors are constructed using molecular tools. These “hybrid” vectors are designed by combining different components from various origins (bacteriophage, F plasmid etc) to create a capacity to load larger insert size and higher transfection efficiency.

1-4.2 Phage Vectors:

To insert DNA fragments of more than 10 kb, normally plasmids are not the suitable vehicles, as large inserts may trigger plasmid rearrangement or affect plasmid replication. This leads to development of a new class of vectors based on bacteriophages. Amongst various bacteriophages available such as λ, T4, T5, and T7 phages; the λ phage gained favourable attention due to its unique life cycle.

λ phage

Bacteriophage λ contains ~49kb of DNA and has a very efficient mechanism for delivering its genome into a bacterium. Two key features contribute to its utility as a vector to clone larger DNA fragments:

1. One-third λ genome is nonessential and could be replaced with foreign DNA. Approximately 24.6kb of λ genome can be deleted, hence maximum insert size could be upto 26 kb.

2. Packing of DNA in phage could only take place if the size is between 40 and 52 kb long, a constraint that can be used to ensure packaging.

Two problems had to be addressed before λ -based cloning vectors could be developed:

•  The size limitation of the insert is determined by the genome size of phage λ (distance between the cos sites). The size range of the modified genome size should be within the range between 78-105% of the genome size for proper packaging. If >2.4kb is inserted to full length λ vector, the packaging efficiency is reduced. Hence λ vector should be smaller in size than wild type λ genome.

•  The large λ genome has a few unique recognition sequences for bacterial restriction endonuclease. Bacterial restriction digestion system may target the modified vector and cleave the λ DNA molecule. This limitation can be overcome by replacing or mutating the restriction sites.

Two types of vector have been developed using λ genome: