Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences. Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs.

In the laboratory, restriction enzymes (or restriction endonucleases) are used to cut DNA into smaller fragments. The cuts are always made at specific nucleotide sequences.

Restriction enzymes

As illustrated in Figure 1, key steps in the workflow of nucleic acid gel electrophoresis are:

1. Selecting and preparing gels. Agarose gels. Polyacrylamide gels. Buffer choice in gel preparation.
2. Preparing standards and samples. Nucleic acid ladder selection. Sample and ladder preparation. Loading dye and buffer choice.

Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. This is a big part of why PCR is an important tool: it produces enough copies of a DNA sequence that we can see or manipulate that region of DNA.

What is PCR. The polymerase chain reaction (PCR) is a biochemical technology used to amplify a specific DNA target in a test tube. In other words, it is a technology that both can find a “needle in a haystack” and then make billion of identical copies of the “needle”.

The methods used for the detection of DNA are:

1. UV- Vis Spectrophotometric analysis.
2. Fluorometric analysis.
3. DNA Precipitation.
4. DNA Gel electrophoresis.
5. Polymerase chain reaction.

Among the techniques we use are:

• DNA sequencing – to identify bacteria, moulds and yeasts.
• Riboprinter analysis – for bacterial identification and characterisation.
• Repeat–based polymerase chain reaction – for assessing the similarity of microorganisms.
• Rapid pathogen confirmation by polymerase chain reaction.

Summary

• Physical control includes such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration.
• Chemical control refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.

Many viral diseases are controlled by reducing exposure to the virus by (1) eliminating nonhuman reservoirs, (2) eliminating the vector, and (3) improving sanitation.

Protozoan cysts are the hardest to kill, with Cryptosporidium being harder to kill than Giardia.

Sterilization: the removal or destruction of all living microorganisms. Heating is the most common method used for killing microbes, including the most resistant forms, such as endospores.

How to kill Bacteria?

• How to kill Bacteria?
• The process of killing bacteria and other micro-organisms either in a vegetative or a spore state is known as sterilization.
• Sterilization or bacterial killing is brought about by many methods, such as physical methods, irradiation and chemical agents or disinfectants.

The only way to kill bacteria by temperature is by cooking food at temperatures of 165 degrees or more. Bacteria also die in highly acidic environments like pickle juice.

Even without antibiotics, most people can fight off a bacterial infection, especially if symptoms are mild. About 70 percent of the time, symptoms of acute bacterial sinus infections go away within two weeks without antibiotics.

Seven best natural antibiotics

1. Garlic. Cultures across the world have long recognized garlic for its preventive and curative powers.
2. Honey. Since the time of Aristotle, honey has been used as an ointment that helps wounds to heal and prevents or draws out infection.
3. Ginger.
4. Echinacea.
5. Goldenseal.
6. Clove.
7. Oregano.

Restriction enzyme: An enzyme from bacteria that can recognize specific base sequences in DNA and cut the DNA at that site (the restriction site). A restriction enzyme acts as a biochemical scissors. Also called a restriction endonuclease.

There are two major types of nucleases: (1) exonucleases and (2) endonucleases. Exonucleases are capable of removing nucleotides one at a time from a DNA molecule whereas endonucleases work by cleaving the phosphodiester bonds within DNA molecule.

DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair.

The enzyme EcoRI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides. This staggered cut leaves a pair of identical single-stranded “sticky ends.” The ends are called sticky because they can hydrogen bond (stick) to a complementary sequence.

Type I toposiomerase is monomeric structured enzyme found in mammalian cells. This enzyme binds double-strand DNA and prevents torsional stress during replication or transcription cleavage of single strand DNA. Topoisomerase I inhibitors are mainly used in the treatment of colorectal and ovarian cancers.

Topoisomerase inhibitors in current use in the United States include irinotecan and topotecan, inhibitors of topoisomerase I, and etoposide and teniposide, inhibitors of topoisomerase II. All four agents are semisynthetic analogues of natural toxins that were initially identified in plants.