Gene Cloning

102 Gene Cloning
parB
Cm r
parA
repE
SP6
oriS
T7
BamHI
Figure 4.11 The main features of a typical bacterial artificial
chromosome (BAC). The oriS and repE genes derived from the E. coli fertility
or F plasmid, are required for replication. The partitioning genes parA and parB
are required to ensure that each daughter cell receives a copy of the plasmid
after cell division. BACs usually contain a selectable marker, in this case
Chloramphenicol resistance (Cm r ). T7 and SP6 are universal promoters to ensure
gene expression from the cloned fragment. In the example shown large DNA
fragments can be cloned between the two BamHI sites.
number of problems, however, if you are working with eukaryotes (Section
4.8). For example, for a eukaryotic organism, particularly one with a large
genome, you may need a prohibitively large number of clones to make a
representative library (Table 4.2). Genomic libraries also contain all the
untranslated sequences including introns; if you need your protein to be
expressed in E. coli you will need clones without introns.
An alternative approach, when you want to find a clone carrying your
gene of interest, instead of making a library from the entire genome of an
organism, is to make one from the mRNA. This will include only those parts
of the genome that are expressed in the particular cell type from which the
mRNA is prepared, and will contain the coding regions for eukaryotic
Table 4.2. Number of clones required for a representative library in different types
of vector
Vector Plasmid Bacteriophage λ Cosmid BAC YAC
Insert Size (20 kb) (25 kb) (45 kb) (300 kb) (1 Mb)
E. coli 1,066 ,852 ,473 ,69 ,19
Yeast 2,761 2,208 1,226 ,182 ,53
Human 757,318 605,854 336,584 50,486 15,144
Rice 96,706 77,365 42,979 6,445 1,932
The table shows the number of clones required to make a library, with a 99% chance of obtaining any
particular fragment, from a range of organisms of differing genome size; the values have been calculated using
the formula given in section 4.4.