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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