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9.2 Cloning DNA Fragments 325

behave like normal chromosomes when introduced into a Figure 9.5 Cloning recombinant DNA molecules.
host cell. A bacterial artificial chromosome (BAC) can ac- (a) Recombinant DNA construction. Cutting genomic DNA with a
commodate a DNA insert of 300 kb. Yeast artificial chro- restriction enzyme produces many fragments, each of which can
mosomes (YACs) can incorporate even larger DNA inserts form a different recombinant DNA molecule. (b) Obtaining clones
of bacterial cells containing recombinant plasmids. Recombinant
up to 2000 kb (2 Mb). In addition to their use in molecular DNAs [from part (a)] are added to ampicillin-sensitive E. coli cells.
cloning, YACs can help investigators analyze functional Only cells transformed with recombinant plasmids (or more rarely
elements of chromosomes such as centromeres; we will with a religated vector lacking a foreign DNA insert) will grow on
thus discuss YACs in greater detail in Chapter 12 on The a petri plate containing ampicillin. Each colony on the plate contains
Eukaryotic Chromosome. millions of identical descendants from a single bacterial cell
transformed with a single recombinant DNA molecule.
(a) Constructing recombinant DNA molecules.
Host Cells Take Up and Amplify Human DNA Plasmid vectors
Recombinant DNA
EcoRI site EcoRI site
Although each type of vector functions in a slightly different
way and enters a specific kind of host, the general scheme of
entering a host cell and taking advantage of the cellular envi- Origin of
ronment to replicate itself is the same for all. Figure 9.5 il- Human DNA and replication
lustrates how scientists obtain E. coli cells that contain plasmid vectors
are cut with EcoRI.
recombinant DNA molecules in which human DNA frag- Gene for ampicillin
resistance
ments were ligated into a plasmid vector. The procedure
starts with vector and human genomic DNAs cut with the DNA ligase
same restriction enzyme, which are then mixed together in
the presence of DNA ligase to create hundreds of thousands
of different recombinant DNAs, each with a different frag-
ment of the human genome (Fig. 9.5a). Researchers must
then introduce these molecules into E. coli such that each
cell contains only a single type of recombinant DNA.

Transformation of host cells
Transformation, as you saw in Chapter 6, is the process by
which a cell or organism takes up a foreign DNA molecule, Rec DNA l
Recombinant DNA moleculesmoleculeslombinant DNAi
changing the genetic characteristics of that cell or organ-
ism. What we now describe is similar to what Avery and (b) Transforming E. coli cells with recombinant DNAsmbinant DNAsng E. coli cells with EE recom
his colleagues did in the transformation experiments that
determined DNA was the molecule of heredity (recall
Fig. 6.4), but the method outlined here is more efficient. Host chromosome
Recombinant DNA molecules are first added to a sus-
pension of specially prepared E. coli that are sensitive to Plasmid
the antibiotic ampicillin. Under conditions favoring entry,
such as suspension of the bacterial cells in a cold CaCl 2
solution or treatment of the solution with high-voltage
electric shock (a technique known as electroporation), the
plasmids will enter about 1 in 1000 cells (Fig. 9.5b). These
protocols increase the permeability of the bacterial cell E. coli plated onto medium containing
membrane, in essence punching temporary holes through ampicillin. Only cells containing
which the DNA gains entry. The probability that any one plasmids are able to grow.
plasmid will enter any one cell is so low (0.001) that the
probability of simultaneous entry of two plasmids into a
single cell is insignificant (0.001 × 0.001 = 0.000001). nutrients, and ampicillin. Only cells transformed by a plas-
mid providing resistance to ampicillin will be able to grow
and multiply in the presence of the antibiotic (Fig. 9.5b). The
Identification and isolation of transformed cells plasmid’s origin of replication enables it to replicate in the
To identify the 0.1% of cells housing a plasmid, the bacteria- bacterial cell independently of the bacterial chromosome; in
plasmid mixture is decanted onto a plate containing agar, fact, most plasmids replicate so well that a single bacterial