274 7.4  Molecular Cloning

7.4.1  CLONING BASICS

Molecular cloning describes a suite of tools using a combination of genetic engineering,

cell and molecular biology, and biochemistry to generate modified DNA to enable it to be

replicated within a host organism (“cloning” simply refers to generating a population of cells

all containing the same DNA genetic code). The modified DNA may be derived from the

same or different species as the host organism.

In essence, for cloning of genomic DNA (i.e., DNA obtained from a cell’s nucleus), the

source DNA, which is to be modified and ultimately cloned, is first isolated and purified from

its originator species. Any tissue/​cell source can, in principle, be used for this provided the

DNA is mostly intact. This DNA is purified (using a phenol extraction), and the number of

purified DNA molecules present is amplified using polymerase chain reaction (PCR) (see

Chapter 2). To ensure efficient PCR, primers need to be added to the DNA sequence (short

sequences of 10–​20 nucleotide base pairs that act as binding sites for initiating DNA repli­

cation by the enzyme DNA polymerase). PCR can also be used on RNA sample sources, but

using a modified PCR technique of the reverse transcription polymerase chain reaction that

first converts RNA back into complementary DNA (cDNA), which is then amplified using

conventional PCR. A similar process can also be used on synthetic DNA, that is, artificial

DNA sequences not from a native cell or tissue source.

The amplified, purified DNA is then chemically broken up into fragments by restriction

endonuclease enzymes, which cut the DNA at specific sequence locations. At this stage, add­

itional small segments of DNA from other sources may be added that are designed to bind

to specific cut ends of the DNA fragments. These modified fragments are then combined

with vector DNA. In molecular biology, a vector is a DNA molecule that is used to carry

modified (often foreign) DNA into a host cell, where it will ultimately be replicated and the

genes in that recombinant DNA expressed can be replicated and/​or expressed. Vectors are

generally variants of either bacterial plasmids or viruses (see Chapter 2). Such a vector that

contains the modified DNA is known as recombinant DNA. Vectors in general are designed

to have multiple specific sequence restriction sites that recognize the corresponding fragment

ends (called “sticky ends”) of the DNA generated by the cutting action of the restriction

endonucleases. Another enzyme called “DNA ligase” catalyzes the binding of the sticky ends

into the vector DNA at the appropriate restriction site in the vector, in a process called liga­

tion. It is possible for other ligation products to form at this stage in addition to the desired

recombinant DNA, but these can be isolated at a later stage after the recombinant DNA has

been inserted in the host cell.

KEY POINT 7.4

The major types of vectors are viruses and plasmids, of which the latter is the most

common. Also, hybrid vectors exist such as a “cosmid” constructed from a lambda

phage and a plasmid, and artificial chromosomes that are relatively large modified

chromosome segments of DNA inserted into a plasmid. All vectors possess an origin

of replication, multiple restriction sites (also known as multiple cloning sites), and one

or more selectable marker genes.

Insertion of the recombinant DNA into the target host cell is done through a process

called either “transformation” for bacterial cells, “transfection” for eukaryotic cells, or, if a

virus is used as a vector, “transduction” (the term “transformation” in the context of animal

cells actually refers to changing to a cancerous state, so is avoided here). The recombinant

DNA needs to pass through the cell membrane barrier, and this can be achieved using both

natural and artificial means. For natural transformation to occur, the cell must be in a spe­

cific physiological state, termed competent, which requires the expression of typically tens of

different proteins in bacteria to allow the cell to take up and incorporate external DNA from

solution (e.g., filamentous pili structures of the outer member, as well as protein complexes