Choosing a cloning vector

DNA libraries are stratified by type of genome as well as type of vector. Classification by type of vector is equivalent to classification by size. There are libraries which contain the entire chromosomes (YAC libraries) as well as the libraries containing pieces of 30kb long.

Vectors must be relatively small molecules for convenience of manipulation. they must be capable of prolific replication in a living cell, thereby enabling the amplification of the inserted donor fragment. Another important requirement is that there must be convenient restriction sites that can be used for insertion of the DNA to be cloned. Generally, we would like to see a unique restriction site because then the insert can be specifically targeted to one site in the vector. It's also desirable that there be a mechanism for easy identification, recovery and sequencing of recombinant molecule. There are numerous cloning vectors in current use and the choice between them usually depends on the size of DNA fragment to be cloned.

The following is a (incomplete) list of vectors which are being used for library creation.

1. Bacteriophage M13
   M13 is extensively used by LLNL in the context of shot-gun sequencing (see below). The size of inserts is about 1,500 bps, i.e. the fragments grown in M13 are ready to be sequenced. M13 contains single--stranded inserts, i.e. there is not going to be a denaturing step in preparation of M13 insert for sequencing.

   The disadvantage of this particular vector is a large cloning bias. M13 is prone to loosing (refusing to amplify with) certain types of sequences. I.e. if only M13 were to be used, certain sequences in genome would never be discovered.

2. Plasmids
   Plasmids are characterized by ability to replicate prolifically. They produce double--stranded fragments. The typical size of insert is about 3,500 bps long. In theory plasmids can hold up to 20kb; however, they easily lose inserts of such size.

3. -phage
   -phages can accept inserts of about 10-15 kb long and is characterized by good ``packaging'' in the sense that it's very unlikely to lose its insert.

4. Cosmids
   This an artificial vector (note that both, plasmids and --phages had been artificially modified as well). The idea behind its creation was to combine ``good'' properties of plasmids and --phages. In particular, cosmids rarely lose inserts, replicate intensely and, in addition, can hold inserts of about 45kb in size. There are extensive cosmid libraries created.

5. YAC -- Yeast Artificial Chromosome
   These artificial vectors in amplified in yeast and hence is the name. YACs can hold huge inserts, up to and beyond 1,000kb, i.e. the entire chromosomes are being replicated in this vector. However, the problem with YAC libraries is that they contain up to if chimeras and it's extremely laborious to separate chimeras from ``real'' recombinants. (A chimera is a recombinant molecule in which non-contiguous donor fragments are being joined together.)

6. BAC -- Bacterial Artificial Chromosome
   BACs are amplified in bacteria as the name suggests. BACs can hold inserts of up to 300kb; however, the average size is about 100kb. BACs inserts can be manipulated with standard plasmid technology and they form fewer chimeras than YACs.