Principles of Plant Genetics and Breeding

232 CHAPTER 14
called a transgenic cultivar or a genetically modified
(GM) cultivar. Generally, an organism developed by
the rDNA procedure is called a genetically modified
organism (GMO).
Certain basic steps are common to all rDNA projects:
1 The DNA of interest that is to be transferred (the
transgene) is extracted from the source organism.
The specific DNA sequence of interest is cut out
using special enzymes.
2 The transgene is inserted into a special DNA
molecule (a cloning vector) and joined to produce a
new rDNA molecule.
3 The rDNA is transferred into and maintained in a
host cell (bacterium) by the process of transformation.
The vector replicates, producing identical
copies (called clones) of the insert DNA.
4 The host cells with the cloned transgene are identified
and isolated from untransformed cells.
5 The cloned transgene can be manipulated such that the
protein product it encodes is expressed by a host cell.
Landmark discoveries in the application of
molecular biology
Numerous discoveries have been made over the years
that plant breeders and other researchers may use for
manipulating plants. An overview of the most influential
tools is given here.
Restriction enzymes
The gene of interest must first be excised from the
source genome before it can be transferred into another
genome. The most common method of cutting DNA is
by using special bacterial enzymes called restriction
endonucleases (or simply restriction enzymes). These
enzymes are base-specific and cut between specific DNA
bases after identifying a short sequence of bases unique
to the enzyme (called the recognition sequence or site).
An example is 5′ . . . TCGA ...3′ for the bacterium
Thermus aquaticus (only one chain is shown).
Southern hybridization
Southern hybridization (or Southern blotting) was
discovered by E. Southern as a technique for isolating a
specific DNA from a mixture of fragments following a
restriction enzyme digest. The DNA digest is denatured
into single strands. The fragments are transferred to a
DNA-binding material (nylon or nitrocellulose), a pro-
cess called blotting. The blot is exposed to a solution
containing a labeled probe (a nucleotide sequence that
is complementary to the DNA sequence of interest) to
bind (hybridize) to it, thereby positively identifying the
gene or DNA sequence of interest.
Reverse transcriptase
As discussed in Chapter 3, the genetic information of
the DNA is transcribed into a template (mRNA) by an
enzyme called DNA transcriptase, for onward translation
into protein. The discovery of the enzyme reverse
transcriptase was a major breakthrough in molecular
biology because it allowed researchers to synthesize a
complementary copy of a given DNA (called complementary
DNA or cDNA). Researchers, knowing the
protein product, can now work backwards to synthesize
its DNA.
Polymerase chain reaction
The polymerase chain reaction (PCR) is an enzymatic
amplification of a DNA fragment. This discovery, among
other applications, allows researchers to make millions
of copies of DNA from just one copy. In its basic
form, the PCR technique uses enzymes to amplify a
DNA fragment by flanking it with two oligonucleotide
primers that hybridize to opposite strands of the target
sequence. A basic PCR cycle consists of three stages:
(i) heat denaturation (at about 95°C) to separate the
double strands of the target DNA; (ii) renaturation at a
cooler temperature (about 55°C) to allow the primers
to anneal to their complementary sequence in the
single-stranded target DNA; and (iii) extension (at
about 75°C) of the primers to create copies of the
target DNA. The PCR mixture consists of DNA polymerase
and the four DNA nucleotides, along with the
source DNA. The PCR occurs in an automated unit
called a thermocycler.
The technique can also be used where only one
sequence suitable as a primer-binding site is known
(called anchored PCR), or to amplify a region of DNA
of unknown sequence that flanks a known sequence
(called inverse PCR). There are modifications of the
PCR (e.g., real time or RT-PCR).
Rapid sequencing
DNA sequencing allows researchers to determine the
complete order (sequence) of bases in a DNA molecule.
Sequencing provides information on gene organization