Cloning vectors

Diagram of a cloning vector capable of autonomous replication in S. pombe, S. cerevisiae and E. coli

1. Plasmids

Useful vectors must contain a cloning site, a replicator, and a selectable marker
A very large number of chimeric plasmid vectors, containing DNA segments from a variety of naturally occurring plasmids, plus some man-made sequences, is available. Many plasmid vectors have been designed for specific purposes.
pBR322--most widely used; moderately high copy number; variety of cloning sites; insertions can be SELECTED based on loss of resistance to ampicillin or tetracycline
pUC18--introduced by Joachim Messing; higher copy number than pBR322 due to loss of the regulatory protein rop; MULTIPLE cloning site; blue-white selection
Plasmids with phage RNA polymerase promoters
Plasmids designed for protein expression (a TYPE of expression vector) in E. coli or in other cell types
- usually contain cloning sites within open reading frames, designed so that the inserted coding sequence will be in proper reading frame
- flanked by promoter and termination signals appropriate for the host organism
Many, many others

2. Lambda vectors

Insert DNA replaces some or all of the genes in the middle third of the lambda genome, which is not essential for lytic growth
Useful for cloning of larger DNA fragments than can easily be cloned in plasmids (up to 24 kbp)
Overall efficiency of cloning and transformation is much higher than for plasmids. Therefore, superior for construction of libraries from large genomes
Generally more difficult to work with than plasmids
Some common vectors are Charon 40 (for very large fragments), lambda EMBL3/lambda EMBL4 (for large fragments), lambda gt10 (efficient cloning of small [0-5 kbp] DNA fragments), and lambda gt11 (expression vector for small DNA fragments; useful for antibody screening of libraries, since many more plaques than bacterial colonies [required for plasmids] can be accommodated on a single plate)

3. Cosmid vectors--features of lambda and plasmids

Like other plasmid vectors, but also contain a cos site from lambda.
Used to clone large DNA fragments (~40 kbp)
Linearized vector DNA and insert DNA are co-ligated into concatamers, then "packaged" into phage heads with a "packaging extract". Wherever two cos sites are separated by 40-50 kbp in the concatamer they, and the intervening DNA, will be excised and packaged into an "infectious" phage that will efficiently inject its DNA contents into a bacterial cell. Once inside the cell, the DNA circularizes by annealing of its cohesive cos ends and then replicates as a large plasmid.
Packaging and "infection" by phage lambda extracts makes cosmids nearly as efficient cloning vectors as lambda itself; suitable for construction of libraries from large genomes

4. P1 vectors--efficient cloning of even larger segments than permitted by cosmids

5. Vectors from filamentous phages--simple production of single-stranded DNA

The filamentous phages (M13, f1, fd) contain a closed circular single-stranded DNA molecule inside their filamentous particles. The ssDNA replicates in 3 stages: SS to RF (double-stranded), RF to RF, and RF to SS. Only the SS form is packaged into phage particles. Infected cells are not killed but efficiently secrete phage particles into the medium.
M13mp vectors
- Developed by Joachim Messing; contain multi-cloning sites and offer blue-white selection
- Some difficulties with obtaining large yields of ds DNA or in maintaining clones with large inserts
"Phagemid" vectors--BlueScript and relatives
- Contain multi-cloning sites, offer blue-white selection, replicate independently as plasmids using the pBR322 origin; contain the M13 (or f1 or fd) replication origin and replicate as filamentous phages, yielding ssDNA-containing phage particles, in the presence of a helper virus.
- Usually contain additional useful features such as inducible promoters for gene expression, and phage RNA polymerase promoters
- Useful for production of single-stranded templates for sequencing or in vitro mutagenesis

6. YACs--helpful for complete coverage of large genomes

Vector requires yeast origin, yeast selectable marker, yeast centromere, yeast telomeres and cloning site(s)
Inserts up to 1.2 Mbp
Low copy number per cell
Possibility of rearrangement during cloning or propagation

7. BACS--also helpful for complete coverage of large genomes

Vector requires E. coli F factor replicon, selectable marker and cloning sites
Inserts up to several hundred kbp
Low copy number per cell
Relatively stable and resistant to rearrangement

You can learn more about BAC vectors from the Caltech Genome Research Laboratory and from the De Jong Laboratory at RPCI.

This page was updated August 30, 1998.