Download restriction enzymes and their uses in specific sequencing to produce

Journal of Pharmaceutical Research And Opinion 1: 5 (2011) 148 – 152.
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REVIEW
RESTRICTION ENZYMES AND THEIR USES IN SPECIFIC SEQUENCING TO PRODUCE
PREDICTABLE FRAGMENT OF DNA MAKING GENETIC ENGINEERING SIMPLY
Nwankwo D.C*, Abalaka M.E.
Department of Microbiology, Federal University of Technology, Minna, Niger State, Nigeria.
ARTICLE INFO
ABSTRACT
Received 4 Sep 2011
Accepted 7 Oct2011
Restriction enzymes are the bedrock for genetic engineering through
which many successful manipulation and modification of DNA fragment by
molecular biologist had been carried out to achieve a desired gene or trait to
enhance human selective evolutions on mature. Restriction enzymes are
protein enzymes that recognize specific nucleotide sequence either in double or
single strand DNA and cleave both strands of DNA containing those sequences
(Robert et al., 1976). Restriction enzymes are discovered during an experiment
to determine the ability of a bacteriophge (the name given to viruses that infect
bacteria) to infect two different strains of Escherichia coli strain B and strain K
in 1970. Such enzymes, found in bacteria and archaea, are thought to have
evolved to provide a defense mechanism against invading viruses (Arber and
Linn, 1969). Inside a bacterial host, the restriction enzymes selectively cut up
foreign DNA in a process called restriction; host DNA is methylated by a
modification enzyme (a methylase) to protect it form the restriction enzyme;s
activity. Collectively, these two processes form the restriction modification
system. To cut the DNA, a restriction enzyme makes two incisions, once
through each sugar-phosphate backbone (double helix).
The first restriction enzymes to be isolated are HmdII (Danna and
Nathans, 1971). There are different kinds of restriction enzymes, but
specifically there are four classes of restriction enzymes endnuclease such as.
Type I, II and III respectively including artificial restriction enzymes with
specific functions. Each enzyme is named after the bacterium from which it was
isolated using the naming system based on bacterium genus, species and
strains.
They are used on different scientific applications in genetic engineering
that involves production of human hormone in medicine, isolation of
predictable fragment of DNA through shotgun method, cloning of desired
fragment of DNA through recombinant technique, restriction fragment length
polymorphism (RFLP) analysis, and Southern blotting methods, amplifying
DNA fragment through PCR method, and production of molecular husbandry.
Through these techniques desired genes had been produced, making genetic
engineering profitable and simply
Corresponding Author:
Nwankwo D. C.
Department of Microbiology,
Federal University of
Technology, Minna, Nigeria
State.
[email protected]
KeyWords
Restriction
enzymes, DNA, endonuleases,
©2011, JPRO, All Right Reserved.
INTRODUCTION
Recognition site
Restriction enzymes recognize a specific sequence
of nucleotides and produce a double-stranded cut in the
DNA. While recognition sequences very between 4 and 8
nucleotides, many of them are palindromic, which
correspond to nitrogenous base sequences that read the
backwards and forwards. In theory, there are two types of
palindromic sequences that can be possible in DNA. The
mirror-like palindrome is similar to those found in
ordinary text, in which a sequence reads the same forward
and backwards on the same DNA strand (i.e., single
stranded) as in GTAATG. The inverted repeat palindrome is
also a sequence that reads the same forward and
backwards, but the forward and backward sequences are
found in complementary DNA strands (i.e., double
stranded) as in GTATAC i.e GTATAC complementary to
CATATG (David, 2005). Recognition sequences in DNA
differ for each restriction enzyme, producing differences in
the length, sequence and strand orientation (‘5 end or the
3’ end) of a sticky-end “overhang” of an enzyme restriction
(Goodsell, 2002).
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Nwankwo et.al / Restriction Enzymes and their Uses in Specific Sequencing to Produce Predictable Fragment of DNA
making Genetic Engineering Simply
Different restriction enzymes that recognize the
same sequence are known as neoschizomers. These often
cleave in a different locales of the sequence; however,
different enzymes that recognize and cleave in the same
location are known as an isoschizomer.
Types of restriction endonuleases
Restriction endonuleases are classified into three
or four groups (Types I, II and III) based on their
composition and enzyme cofactor requirements, the nature
of their target sequence, and the position of their DNA
cleavage site relative to the target sequence, and the
position of their DNA cleavage site relative to the target
sequence. There are four classes of restriction
endonucleases: types I, II, III and IV. There are different
enzymes that recognize specific short DNA sequences and
carry out the endonucleolytic cleavage of DNA to give
specific double-stranded fragments with terminal
phosphates. They differ in their recognition sequence,
submit composition, cleavage position, and cofactor
requirements.
There are different enzymes
The first to be identified and are characteristic of
two different strains (K-2 and B) of E. (Murray 2001) these
enzymes cut at a site that differs, and is some distance (at
least 1000 bp) away, from their recognition site. The
recognition site is asymmetrical and is composed to two
portions-one containing 3-4 nucleotides, and another
containing 4-5 nucleotides-separated by a spacer of about
6-8 nucleotides. Several enzyme cofactors, including SAdenosyl methionine (AdoMet). Hydrolyzed adenosine
triphosphate (ATP), and magnesium (Mg2+) ions, are
required for heir activity. Type I restriction enzymes posses
three subunits called HsdR, HsdM, and HsdS; HsdR is
required for restriction; HsdM is necessary for adding
methyl groups to host DNA (methyltransferase activity)
and HsdS is important for specificity of cut site recognition
in addition to its methltranferase activity.
Typical type II restriction enzymes differ from type
I restriction enzymes in several ways. They are a dimer of
only one type of subunit; their recognition site and they do
not use ATP or AdoMet for their activity-they usually
require only Mg2+ as a cofactor. These are the most
commonly available and used restriction enzymes. In the
1990s and early 2000s, new enzymes from this family were
discovered that did not follow all the classical criteria of
this enzyme class, and new subfamily nomenclature was
eveloped to divide this large family into subcategories
based on deviations from typical characteristics of type II
enzymes. Type IIB restriction enzymes (e.g BcgL and bpll)
are multimers, containing more than one subunit (Pingoud
and Jeltsch, 2001) they cleave DNA on both sides of their
recognition to cut out the recognition site.
They require both AdoMet and Mg2+ cofactors. Type IIE
restriction endonucleases (e.g Nael) cleave DNA following
interaction with two copies of their recognition sequence.
One recognition site acts as the target for cleavage, while
the other acts as an allosteric effector that speeds up or
improves the efficiency of enzyme cleavage. Similar to type
IIE enzymes, type IIF restriction endonucleases (e.g
NgoMIV) interact with two copies of their recognition
sequence but cleave both sequences at the same time. Type
IIG restriction endonucleases (Eco571) do have a single
subunit, like classical type II restriction enzymes, but
require the cofactor AdoMet to be active. Type IIM
restriction endonucleases, such as Dpnl, are able to
recognize and cut methylated DNA (Pingoud and Jeltsch,
2001). Type IIS restriction endonucleases (e.g Fokl) cleave
DNA at a defined distance from their non-palindromic
asymmetric recognition sites. These enzymes may function
as dimmers. Type IIT restriction enzymes (e.g Bpu10I and
BsII) are composed of two different subunits. Some
recognize palindromic sequences while others have
asymmetric recognition sites (Pingoud and Jeltsch, 2001).
Type III
Type III restriction enzymes (e.g EcoP15)
recognize two separate non-palindromic sequences that
are inversely oriented. They cut DNA about 20-30 base
pairs after the recognition site. These enzymes contain
more than one subunit and require AdoMet and ATP
cofactors for their roles in DNA metylation and restriction,
respectively (Meisel et al., 1992)
Artificial restriction enzymes
Artificial restriction enzymes can be generated by
fusing a natural or engineered DNA bindings domain to a
nuclease domain often the cleavage domain of the type IIS
restriction enzyme Fokl (Kim et al., 1996). Such artificial
restriction enzymes can target large DNA sites (up to 36
bp) and can be engineered to bind to desired DNA
sequences. Zinc finger nucleases are the most commonly
used artificial restriction enzymes and are generally used
in genetic engineering applications, but can also be used for
more standard gene cloning applications. Other artificial
restriction enzymes are based on the DNA binding domain
of TAL effectors (Huang et al., 2010).
Applications of restriction enzymes in genetic
engineering
They are used to assist insertion of genes into
plasmid vectors during gene cloning and protein
expression experiments. For optimal use, plasmids that are
commonly used for gene cloning are modified to include a
short polylinker sequence (called the multiple cloning site,
or MCS) rich in restriction enzyme recognition sequences.
This allows flexibility when inserting gene fragments into
the plasmid vector; restriction sites contained naturally
within genes influence the choice of endonuclease for
digesting the DNA since it is necessary to avoid restriction
of wanted DNA while intentionally cutting the ends of the
DNA. To clone a gene fragment into a vector, both plasmid
DNA and gene insert are typically cut with the same
restriction enzymes, and then glued together with the
assistance of an enzyme known as a DNA ligase (Russel et
al., 2001).
Restriction enzymes can also be used to
distinguish gene alleles by specifically recognizing single
base changes in DNA known as Single Nucleotide
Polymorphisms (SNPs) (Zhang et al., 2005). This is only
possible if a SNP alters the restriction site present in the
allele. In this method, the restriction enzyme can be used to
genotype a DNA sample without the need for expensive
gene sequencing. The sample is first digested with the
restriction enzyme to generate DNA fragments, and then
the different sized fragments separated by gel
electrophoresis. In general, alleles with correct restriction
sites will generate two visible bands of DNA on the gel
electrophoresis. In general, alleles with correct restriction
sites will generate two visible bands of DNA on the gel, and
those with altered restriction sites will not be cut and will
generate only a single band. The number of bands reveals
the sample subject’s genotype, e.g restriction mapping.
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making Genetic Engineering Simply
The concept of introducing specific trait through the
isolation and manipulation of DNA has been enhance by
restriction enzymes, in recent time, genetic engineering
had changed from a term characterizing the selection of
desired traits to a concept describing the isolation,
modification and introduction of genes encoding the
desired traits. This means that genes can be conceived of as
molecular machines capable of being designed to modify
their performance. This means that genes can be conceived
of as molecular machines capable of being designed to
modify their performance. This concept of genetic
engineering as involving the alteration of specific
nucleotide sequence of DNA was made possible by a virtual
explosion in understanding the gene structure and their
functions.
The result of a desired gene for a specific trait had
led to the development of successive approaches to achieve
the desired genetic engineering goals. These approach can
be thought as follows:
Recombinant DNA cloning
One of the keystones of recombinant DNA cloning
was the intriguing observation that a bacterial strain could
be relatively resistant to infection by a bacteriophage stock
grown on a different but closely related bacterial strain.
The infrequent infection event that occurred, however,
could give rise to a bacteriophages stock adapted to
efficient infection of the second bacterial strain, but no
longer able to efficiently infect the original host (12). This
observation ultimately resulted in the discovery and
characterization of restriction enzymes, naturally occurring
enzymes that recognize and cleave DNA in a site-specific
manner.
Because these enzymes recognize and cleave at
specific sequence along a DNA molecule (Robert, 1984),
they allowed conversion of a mixture of randomly sheared
chromosomal DNA fragments to a set of DNA fragments
with sizes determined by the location of restriction
cleavage sites within the DNA molecules. For small
genomes, such as those of bacteriophages, plasmids, and
viruses, this innovation alone was sufficient to allow the
physical characterization of DNA molecules and the
correlation of certain genetic traits with specific DNA
fragments. However, analysis of DNA restriction fragments
alone was not sufficiently powerful to allow efficient
analysis of larger genomes.
The second critical innovation that allowed the
evolution of second generation DNA technology was the
concept of using small naturally occurring replicon as
molecular vehicles (Boyer et al., 1974). Or vectors, to allow
propagation and biological amplification of specific DNA
fragments (Thomas et al., 1974). Restriction digestion of
DNA followed by mixture with a vector DNA molecule
allowed the annealing of the cohesive restriction termini to
cause the formation of new, recombinant DNA molecules.
When the nicks in the annealed termini were sealed with
DNA ligase, either in vitro prior to or in vivo after insertion
of the recombinants into a host, each resulting construct
allowed whatever DNA fragment happened to have been
ligated to the vector to be isolated by cloning the host
organism. This innovation circumvented the need to
amplify the entire genome of an organism to obtain
workable amounts of a specific gene an individual gene
could be amplified by growing a recombinant DNA
molecule in a bacterial clone. This simple and useful form
of gene engineering provided the foundation for early
transgenic technology that used extra chromosomal
vectors for the overproduction of recombinant proteins in
bacteria.
The ability to specifically cleave, biologically
amplify and isolate specific DNA fragments greatly
enhanced the efficiency of construction and isolation of a
desired DNA molecule and gave rise to the recombinant
DNA technology. It provided the means to take molecular
machines apart and to rebuild them to suit human needs.
When combined with improved screening technologies,
DNA sequence analysis and DNA synthesis, the
recombinant DNA technology, give rise to an avalanche of
information concerning gene structure and function.
In spite of the tremendous power afforded by this
technology, recombinant DNA technology still suffered
from key limitations when applied to many specific genetic
engineering goals. The ability to make desired recombinant
molecules was often dependent on the fortuitous
occurrence of restriction cleavage sites. Even in cases
where construction of a specific construct was technically
feasible, finding the desired molecule amongst a
background undesired molecules might require exhaustive
screening. In addition, the need to amplify the desired DNA
fragment on a vector in a host at times resulted in the
generation of deletions, insertions or rearrangement in the
cloned DNA fragment. The presence of some recombinants
has proven to be detrimental to survival of the host, and a
specific host will not necessarily propagate all
recombinant. Once the desired DNA fragment has been
found, the same exhaustive screening must be repeated if
the same gene is required from a different organism. For
instant, the requirement for a biological host, one of the
intrinsic components of the second generation DNA
technology can become a significant obstacle to application
of recombinant DNA cloning technology certain genetic
engineering problems.
Polymerization chain reaction (PCR) DNA base
manipulation in-vitro
The invention of PCR (Mullis et al., 1986) led to
another fundamental change in the way genetic material
can be analyzed and exploited. Recombinant DNA cloning
approaches are to a great extent dependent on the
availability of enzyme recognition sites within the DNA of
interest and on the amplification of the desired DNA
construct by cloning into a host organizer. This approaches
utilize the specificity of chemically synthesized
oligonucliotides and highly efficient in vitro amplification of
DNA to enhance the ability to make desired engineered
DNA molecules. PCR based sequence manipulation
encompasses several applications that, in the recombinant
DNA technology, were typically performed through a
succession of separate techniques, isolation, amplification
and identification of specific gene fragments, as well as sitedirected and random mutagenesis and recombinator, can
be achieved by simple techniques that can often be
completed in a matter of hours.
Many of these techniques are applications of the
principle of overlap extensior (Higuch et al., 1988), a
technique in which the terminus of an amplified sequence
segment is used as a PCR “megaprime” in a subsequent
amplification reactor (Sarkai and Sommer 1990). This
technique provides a method to perform site-directed
mutagenesis on PCR products at an arbitrary distance from
the ends of the molecule and if the mega primer and the
subsequent template are amplified from different genes,
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making Genetic Engineering Simply
then the product is a recombinant molecule that has been
engineered, isolated, and amplified all by the same process.
Other approaches to PCR-based DNA sequence
manipulation do not rely on overlap extensor. These
include ligation of engineered overlaps and engineering of
homologous ends that can undergo homologous
recombination in vivc. Many PCR application; such as using
PCR primers containing type IIS restriction sites of
generate sticky ends of arbitrary sequence (i.e not
including the restriction site itself, which is removed from
the molecule when the strands are cut) to direct the
ligation of PCR products, bridge both recombinant cloning
and PCR technologies. Other approaches that take
advantage of both technologies include the use of
mispriming to introduce or eliminate restriction sites,
generation of cohesive ends by removed of chemically
distinct primers, and generation of cohesive ends by
depriving a proofreading polymerase of certain bases.
May significant development in PCR technology
have had tremendous impact on the versatility of PCR for
sequence manipulator. Problems related to the errors that
may be introduced in the amplification process, for
example were significantly reduced by the description of
high-fidelity reaction condition for the use of Tac
polymerase and by the isolation and production of thermo
stable proofreading polymerases with lower error rates.
Development of conditions that facilitate the amplification
of long segments was also a key to increasing the general
applicability of PCR-mediated DNA manipulator. As with
the recombinant DNA technology, the development of PCRbased technology has been the cumulative result of the
efforts of many researchers in genetic engineering.
Molecular husbandry
The idea of engineering is closely tied to rational
design, a process in which scientific principles are used to
design a thing and them construct it. DNA engineering
technology has made it possible, and in many cases even
simple and economically feasible to create essentially any
gene sequence desired. Many fundamental questions
remaining on this level relate to determining what gene
sequence are desired particularly with respect to protein
engineering a very important application of gene
manipulator. Although existent technology allows the
synthesis of essentially any gene sequence, our current
understanding of the relationships between protein
structure and function does not allow the de novc design of
complex gene products. Many, if not the vast majority, of
genetic engineering projects currently modify a naturally
occurring gene or protein for a practical or experimental
purpose. Natural evolution tends to produce proteins and
other molecules that are optimized for some function in
their cellular environment. Genetic variants become
stabilized in a population by natural selection of beneficial
traits associated with the variant. The history of DNA
engineering by conventional genetics has often involved
the search for naturally occurring genetic variants that
convey a particularly desirable phenotype. Much effort in
pharmacology and other fields has gone into identifying
natural substances that can be used to serve come man
made purpose. Technology has now developed to the point
that many useful or potentially molecules, such as plastics,
are synthetic products that do not normally exist in nature.
Because this, gene and protein models for enzymes to
manipulate these compounds are unlikely to be discovered
as natural genetic variants. Other genetic variants useful for
specialized applications may be detrimenta in vivc, such as
lethal autoantibodie, decreasing the probability of finding
these sequence variants in natural systems.
Linkage genotype with selectable phenotypes is a
key component of establishing genetic variants by natural
selector. This type of linkage, which will likely be a key to
developing genetic variants that do not exist naturally, can
be accomplished experimentally on a molecular basis by
selecting nucleic acid molecules on the basis of some
characteristic determined by their sequence, such as the
ability to bind a target molecule, or by packaging a gene
together with the product with the product it encodes in a
viral particle and selecting the particles based on some
characteristic of the encoded protein. Molecules that bind
to the target of interest can then be amplified, modified and
re-selected, leading to progressively developed phenotype.
This type of approach can be thought of as molecular
evolution.
The development in vitro evolution a sense brings
genetic engineering technology full circle. Random
recombination of PCR products during co-amplification can
be used to perform “sexual PCR”, in which “random” cross
over sat points of homology between co-amplified PCR
products are used an and in vitro approximation to sexual
recombinator. DNA engineering methods provide the
means to use selection analogous to plant or livestock
breeding at the molecular level, as a form of molecular
husbandry in which variants are generated through
mutation and recombination and those that best serve the
desired function, such as affinity to a target, are selected
and propagate. This is essentially genetic breeding of
molecules, and is analogous to old fashioned plant and
animal breeding.
Southern blotting analysis genetic engineering
Southern blot is a techniques routinely used in
molecular biology for detection of a specific DNA sequence
in DNA samples thereby enhancing genetic engineering.
This technique is mainly useful when gene of interest is
rare, and where some of the DNA fragments are larger then
15kb, when DNA molecules are separated on a gel it is
possible to identify molecules
that carry specific
sequences. This identification is accomplished through
the denaturation (separation of individual DNA strands)
and then transfer of the DNA found in the gel to a kind of
paper that is laid against the gel, the DNA now found on the
paper is probed with tagged DNA fragments that, ideally
only bind to specific bands of DNA now found on the
paper. The tagged fragments ( e.g., radioactively tagged)
are then visualized and the location or presence of the DNA
on the original gel is inferred from its location or
presence on the paper.
Restriction fragment length polymorphism analysis
(RFLP)
In the restriction fragment length polymorphism
(RFLP) analysis, the location of restriction sites in a
genome occurs firely randomly, and can differ from person
to person essentially as allelic
difference between
individuals ( in general difference in nucleotide sequences
at specific loci form within population polymorphism, in its
originally meaning implying
differences in the
morphologies of individuals making up a population,
implying genotypic differences). As a result of these
differences between individuals in the location of specific
restriction sites, the distance between the sites varies, this
length of restriction fragments produced by digesting an
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making Genetic Engineering Simply
individuals genome using specific restriction enzymes will
also vary. The variation between individuals is called
restriction fragment length
polymorphism
because
nucleotide sequence of nearly every individual is unique,
RFLPS of each individual are also unique, and thus RFLP
analysis are equally employed to forensically distinguish
individuals (hence the synonymous terms, DNA
fingerprinting).
This technique these days are employed almost in every
population study you can imagine in genetic engineering,
ranging from agriculture to forensics to wild life biology.
CONCLUSION AND RECOMMENDATION
Restriction enzymes as a key
in genetic
engineering techniques has now developed to the point
that the ability to manipulate and modify and synthesize
gene sequence has greatly exceeded basic understanding
of protein gene products. The benefits of restriction
enzymes in genetic engineering can be overemphasized
because they have yielded a wide array of medical,
agricultural and other benefits. One medical benefit is
faster and more abundant production of insulin and in
agriculture, production of hybridized animals.
Recommendations
It is therefore essential that the researchers
should concentrate on those restriction enzymes that will
be more
profitable
in
industries, medicine and
agriculture.
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