Page 25 - Advances in Textile Biotechnology

4 Advances in textile biotechnology

1.1.1 Obtaining, cutting and joining the DNA

Because a DNA cloning strategy initially requires identification of the

genetic sequence coding for a specific protein, the first step is to consider

the source of such a DNA molecule, which represents a specific gene or
portion of a gene. Typical fragments of interest include genomic DNA,
cDNA, polymerase chain reaction (PCR) products and chemically synthe-
sized oligonucleotides.
Two major types of enzymes are crucial for the isolation and construction
of recombinant DNA molecules: restriction endonucleases and DNA
ligases. Restriction endonucleases can be grouped into three classes accord-
ing to their molecular structure, need for specific co-factors, the types of

sequences recognized, and the nature of the cut made in the DNA. We will
focus on type II endonucleases, the most commonly used in recombinant

DNA technology. These enzymes recognize a specific site on double-
stranded DNA, called a restriction site, which comprises a short sequence
of nucleotides, and cleave within it without requiring ATP hydrolysis for
nucleolytic activity (Marx, 1973; Sambrook and Maniatis, 1989). Type II
restriction endonucleases exhibit a high degree of sequence specifi city and
a single base pair change in the recognition site of a restriction endonucle-
ase essentially hinders and blocks enzymatic activity. In contrast, type I and
III restriction endonucleases cause random cleavage patterns in the DNA
which are useless for gene cloning (Gorbalenya and Koonin, 1991). So far,
around 3000 type II restriction enzymes have been identified and only a

small fraction of those has been biochemically characterized. Orthodox
type II endonucleases are composed of two identical polypeptide subunits
that join together to form a homodimer. These homodimers recognize pal-
indromic DNA sequences of 4–8 bp, although 6 bp DNA sequences are the
most recognized by restriction endonucleases used in molecular genetics.
2+
Mg ions are required for catalysis. First, the restriction endonuclease con-
tacts with DNA sugar–phosphate backbone in a non-specific way for effi -

cient recognition of the target site location. Once the restriction site is
located, the recognition process triggers conformational changes in the
enzyme and the DNA, in turn, that leads to the activation of the catalytic

center. In contrast to the non-specific contact, specific binding involves
direct interaction of the enzyme with DNA nitrogenous bases.
Type II restriction enzymes cut the DNA double strand disrupting the
covalent, phosphodiester bond between the phosphate of one nucleotide
and the sugar of an adjacent nucleotide, producing 3′-hydroxyl and 5′-
phosphate ends (detailed reviews of target site location, recognition and
catalysis mechanism of type II endonucleases have been given by Pingoud
and Jeltsch (2001) and Pingoud et al. (2005)).

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