ABSTRACT
The function of restriction endonucleases is mainly
protection against foreign genetic material especially against bacteriophage
DNA. The other functions attributed to these enzymes are recombination and
transposition. Restriction endonucleases make up the restriction-modification
(R-M) systems comprised of endonuclease and methytransferase activities. The
endonuclease recognizes and cleaves foreign DNA on the defined recognition
sites. The methytransferase modifies the recognition sites in the host DNA and
protects it against the activity of endonucleases. The sequences in foreign DNA
are generally not methylated and are subjected to restriction digestion. Each
restriction enzyme recognizes a specific sequence of 4–8 nucleotides in DNA and
cleaves at these sites. According to the description of each reaction in table
1 and table 2; each content was pipetted into the respective labeled tubes
which were then centrifuged, incubated at 37°C and then analyzed using agarose
gel electrophoresis. After, the molecules were run in agarose gel, the trends
for single, double and even triple digestion were observed in L1 through L7 for
plasmid DNA, with L1 as molecular weight marker, L6 and L7 as controls. For mammalian samples, L1-L3 was loaded with
sample DNA whereas L4 served as the control. According to the results observed,
different endonucleases used in this experiment recognized sequences on both
plasmid and mammalian DNA of which they digested with respect to their ability. Problems with enzyme activity can occur under the
following conditions: High glycerol concentration, Enzyme-to-DNA ratio is too
high; pH is too high, Organic solvents, particularly ethanol and any
interference with the DNA molecule. However, by observing the trend of the
bands on the results session and the theory behind this experiment, it was
therefore concluded that the objectives of the practical were achieved, the
activity of the restriction enzymes were demonstrated, digested DNA fragments
were observed after they were allowed to run in agarose gel electrophoresis.
After digestion, enzymes can be mapped on how and where they can cleave on the
DNA sequence.
INTRODUCTION
Restriction endonucleases are enzymes that cleave the
sugar-phosphate backbone of DNA. In most practical settings, a given enzyme
cuts both strands of duplex DNA within a stretch of just a few bases. Several
thousand different restriction endonucleases have been isolated, which
collectively exhibit a few hundred different sequence (substrate)
specificities. A large majority of restriction enzymes have been isolated from
bacteria, where they appear to serve a host-defense role. The idea is that
foreign DNA, for example from an infecting virus, will be chopped up and
inactivated ("restricted") within the bacterium by the restriction
enzyme (Promdonkoy et al, 2003).
Restriction enzymes were discovered in bacteria and
there are now more than 1200 known restriction enzyme types. These enzymes are
named using a simple system. EcoRI, for
example, was isolated from E. coli and
was the first enzyme isolated from a particular strain, hence the designation
of I. HaeIII was isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae,
and was the third enzyme discovered in a particular strain (Li et al, 1991).
The function of
restriction endonucleases is mainly protection against foreign genetic material
especially against bacteriophage DNA. The other functions attributed to these
enzymes are recombination and transposition. Restriction endonucleases make up
the restriction-modification (R-M) systems comprised of endonuclease and
methytransferase activities. The endonuclease recognizes and cleaves foreign
DNA on the defined recognition sites. The methytransferase modifies the
recognition sites in the host DNA and protects it against the activity of
endonucleases. The sequences in foreign DNA are generally not methylated and
are subjected to restriction digestion. Each restriction enzyme recognizes a
specific sequence of 4–8 nucleotides in DNA and cleaves at these sites.
Endonucleases isolated by different organisms with identical recognition sites
are termed isoschizomers (Cheong and Gill, 1997)
Plasmids are used as vectors to clone DNA in bacteria.
One example of a plasmid used for DNA cloning is called pBR322 Plasmid. The
pBR322 plasmid contains a gene that allows the bacteria to be resistant to the
antibiotics tetracycline and amipicillin. To use pBR322 plasmid to clone a
gene, a restriction endonuclease first cleaves the plasmid at a restriction
site. pBR322 plasmid contains three restriction sites: PstI, SalI and ecoRI.
The first two restriction sites are located within the gene that codes for
ampicillin and tetracycline resistance, respectively. Cleaving at either
restriction site will inactivate their respective genes and antibiotic
resistance. The target DNA is cleaved with a restriction endonuclease at the
same restriction site. The target DNA is then annealed to the plasmid using DNA
ligase. After the target DNA is incorporated into the plasmid, the host cell is
grown in an environment containing ampicillin or tetracycline, depending on
which gene was left active. Many copies of the target DNA is created once the
host is able to replicate (Jaurez-perez et
al, 2002).
Another plasmid used as a vector to clone DNA is
called pUC18 plasmid. This plasmid contains a gene that makes the host cell
ampicillin resistant. It also contains a gene that allows it to produce
beta-galactosidase, which is an enzyme degrades certain sugars. The enzyme
produces a blue pigment when exposed to a specific substrate analog. This
allows the host to be readily identified. The gene for beta-galactosidase
contains a polylinker region that contains several restriction sites. The pUC18
plasmid can be cleaved by several different restriction endonucleases which
provide more versatility. When the polylinker sequence is cleaved and the
target DNA is introduced and ligased, this inactivates the gene that codes for
beta-galactosidase and the enzyme will not be produced. The host cell will not produce
a blue pigment when exposed to the substrate analog. This allows the
recombinant cells to be readily identified and isolated (Boonserm et al, 2005).
In this practical, the activity of restriction enzymes
will be demonstrated by setting up restriction digest using different
endonucleases which will either digest the pre-isolated plasmid/mammalian DNA one, two or even three times based on the
ability of the enzyme in reading and recognizing the sequences to cleave on the
DNA molecule . The digested DNA fragments will further be analyzed using
agarose gel electrophoresis (Crickmore et
al, 1998).
METHODS
AND MATERIALS
Table 1: restriction digest of pBR322 DNA with Hind
III, Nde I, Pst I and Sal I
|
Reaction
components and volumes of each
|
Reaction
1
Hind
III
|
Reaction
2
Hind
III + Nde I
|
Reaction
3
Hind
III + Nde I + Pst I
|
Reaction
4
Hind
III + Nde I + Pst I + Sal I
|
Reaction
5
Control
|
|
Nuclease
free H2O
|
14
μl
|
12 μl
|
10 μl
|
8 μl
|
16 μl
|
|
Restriction
enzyme buffer
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
|
Plasmid
DNA
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
|
Restriction
enzyme
|
2 μl
|
2 μl each
|
2 μl each
|
2 μl each
|
0 μl
|
|
Total
volume
|
20
μl
|
20
μl
|
20
μl
|
20
μl
|
20
μl
|
Table 2: restriction digest of pUC18 DNA with Bam III,
Pst I and Bgi I
|
Reaction
components and volumes for each
|
Reaction
1
Bam
III
|
Reaction
2
Pst
I
|
Reaction
3
Bgi
I
|
Reaction
4
Control
|
|
Nuclease
free H2O
|
14 μl
|
14 μl
|
14 μl
|
16 μl
|
|
Restriction
enzyme buffer
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
|
Mammalian
DNA
|
2 μl
|
2 μl
|
2 μl
|
2 μl
|
|
Restriction
enzyme
|
2 μl
|
2 μl
|
2 μl
|
0 μl
|
|
Total
volume
|
20 μl
|
20 μl
|
20 μl
|
20 μl
|
Reactions tubes were obtained and labeled with respect
to the number of reactions to be performed both for table 1 and 2. Following
the sequence as in both table 1 and 2 the components of each reaction were
added to the respective tubes. By pipetting, each content on each tube were
mixed and centrifuged thereafter using a microfuge to collect the contents at
the bottom of the tube. The tubes were then incubated at 37 °C for 1 hour.
After the completion of the digestion, the technique of gel electrophoresis was
used to further analyze the contents on the tubes, this was done by first
adding dye to each tube which were kept in -20°C after incubation, then the
contents were loaded onto agarose gel and allowed to run
RESULTS
The activity of restriction enzymes were observed
after when the sample DNA fragments were allowed to run on an agarose gel
electrophoresis following restriction digestion by various enzymes on both
plasmid and mammalian DNA which were isolated and purified on the early
experiments.
Figure 1: DNA fragments observed after restriction
digest by endonucleases; multiple bands represent triple or double digest while
single bands may either represent a single digestion or no digestion at all.
The left picture also shows RNA contamination on L5-7
DISCUSSION
Restriction enzymes, also known as restriction
endonucleases, are enzymes that cut a DNA molecule at a particular place. They
are essential tools for recombinant DNA technology. The enzyme
"scans" a DNA molecule, looking for a particular sequence, usually of
four to six nucleotides. Once it finds this recognition sequence, it stops and
cuts the strands. This is known as enzyme digestion. On double stranded DNA the
recognition sequence is on both strands, but runs in opposite directions. This
allows the enzyme to cut both strands. Sometimes the cut is blunt; sometimes
the cut is uneven with dangling nucleotides on one of the two strands. This
uneven cut is known as sticky ends. Restriction
enzymes cut at specific sites along the DNA. These sites are determined by the
sequence of bases which usually form palindromes. Palindromes are groups of
letters that read the same in both the forward and backwards orientation. In
the case of DNA the letters are found on both the forward and the reverse strands
of the DNA. For example, the 5’ to 3’ strand may have the sequence GAATTC. The
complimentary bases on the opposite strand will be CTTAAG, which is the same as
reading the first strand backwards! Many enzymes recognize these types of
sequences and will attach to the DNA at this site and then cut the strand
between two of the bases. After gel analysis of the digested fragments of
plasmid DNA, in L1 was observed to have multiple bands since it served as
molecular weight marker. L2 and L3 were observed to have linear single bands
resulting from a single digest by the restriction enzymes. L4 and L5 had multiple
bands resulting from triple digestion, however L5 had RNA contamination as also
observed in L6 and L7 which were the controls. By clearly looking at the
controls L6 and L7 it clear that the samples were digested by the restriction
enzymes, the supercoiled observed in L7 was not observed in any of the samples.
Restriction digestions of plasmid DNA are essential for mapping, cloning and
other recombinant technologies improving molecular biology. For the mammalian
DNA digestion, L1 through L4 only the same kind of band was observed signifying
that there was either a single digestion or no digestion at all. Problems with
enzyme activity can occur under the following conditions: High glycerol
concentration, Enzyme-to-DNA ratio is too high; pH is too high, Organic
solvents, particularly ethanol and any interference with the DNA molecule.
However, by observing the trend of the bands on the results session and the
theory behind this experiment, it was therefore concluded that the objectives
of the practical were achieved, the activity of the restriction enzymes were
demonstrated, digested DNA fragments were observed after they were allowed to
run in agarose gel electrophoresis. After digestion, enzymes can be mapped on
how and where they can cleave on the DNA sequence.
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