4-4.2.2. Cloning of genomic DNA

Various vectors are available for cloning large DNA fragments. λ phage, yeast artificial chromosome, bacterial artificial chromosome etc. are considered as suitable vectors for larger DNA and λ replacement vectors like λDASH and EMBL3 are preferred for construction of genomic DNA library. T4 DNA ligase is used to ligate the selected DNA sequence into the vector.

(1)  λ replacement vectors

The λEMBL series of vectors are widely used for genomic library construction. The multiple cloning sites of these vectors flanking the stuffer fragment contain opposed promoters for the T3 and T7 RNA polymerases. The restriction digestion of the recombinant vector generates short fragments of insert DNA left attached to these promoters. This generates RNA probes for the ends of the DNA insert.These vectors can be made conveniently, directly from the vector, without recourse to sub-cloning.

(2)  High-capacity vectors

The high capacity cloning vectors used for the construction of genomic libraries are cosmids, bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs) and yeast artificial chromosomes (YACs). They are designed to handle longer DNA inserts, much larger than for λ replacement vectors. So they require lower number of recombinantsto be screened for identification of a particular gene of interest.

Vector	Insert size	Features
λ phages	Up to 20-30 kb	Genome size-47 kb, efficient packaging system, replacement vectors usually employed, used to study individual genes.
Cosmids	Up to 40 kb	Contains cos site of λ phage to allow packaging, propagate in E. coli as plasmids, useful for sub-cloning of DNA inserts from YAC, BAC, PAC etc.
Fosmids	35-45 kb	Contains F-plasmid origin of replication and λ cos site, low copy number, stable.
Bacterial artificial chromosomes (BAC)	Up to 300kb	Based on F-plasmid, relatively large and high capacity vectors.
P1 artificial chromosomes (PACs)	Up to 300 kb	Derived from DNA of P1 bacteriophage, combines the features of P1 and BACs, used to clone larger genes and in physical mapping, chromosome walking as well as shotgun sequencing of complex genomes.
Yeast artificial chromosomes (YAC)	Up to 2000kb	Allow identification of successful transformants. (BAC clones are highly stable and highly efficient)

  Table 4-4.2.2. Vectors used for cloning genomic libraries.

The recombinant vectors and insert combinations are grown in E. coli such that a single bacterial colony or viral plaque arises from the ligation of a single genomic DNA fragment into the vector.