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Genetic Engineering

Introduction

Discovery of DNA structure, in the 1950s, represented a huge step in the advancement of biology. However, two decades later, the development of other forms of study and DNA manipulation inaugurated a new era in scientific research: genetic engineering.

Recombinant DNA technology

Technology

Recombinant DNA technology refers to the manipulation of genetic information to create new combinations of DNA into which genes or gene segments from different organisms are integrated. Although in this section we will emphasize the recombination of DNA carried out in the laboratory, we will begin by briefly reviewing nature's strategies for recombinant DNA

Nature

During meiosis I there is a process called cross-linking, in which homologous chromosomes exchange genetic material. This allows each gamete to contain a mixture of the alleles of the original chromosomes, so that both the ovule and he sperm contain recombinant DNA from both parents. This recombinant DNA is inherited to the offspring

Conjugation

Transduction

Transformation

Transformation is the process by which bacteria incorporate segments of DNA that are not their own, capturing them from the environment. The DNA found in environment can come from the genetic material released by another bacterium, for example, upon death. The purpose of transformation is the incorporation of foreign DNA into the chromosome of the recipient bacterium resulting in a stable genetic change

in transduction, viruses that infect bacteria (bacteriophages) transport genetic material from one bacterium to another. Initially, the phage adheres to the bacterial wall, injects its genetic material and takes ad vantage of the entire machine of the cell to replicate it, along with other proteins. From these com ponents, new viral particles are Bumble and will cause rupture Paralysis of the cell subsequently. In this process some viruses, which are called transduction bacteria rophages, encapsulate a por tion of the bacterial DNA.

In the case of conjugation, two bacteria come into contact and one of them transfers genetic material to the other. To do this, a bacterial cell, called a donor, provides the DNA that may be accepted by the recipient cell.

Cloning in laboraty

1) Cut the DNA. Once the gene of interest has been identified, special enzymes will be used to control the DNA in smaller fragments. These enzymes are called red restriction enzymes or restriction endonu cleases. Many of these enzymes are found naturally in bacteria as they are used as a defense mechanism to cut any viral DNA that has been introduced into the cell by a bacteriophage. Restriction enzymes also function as "molecular scissors", which cut a specific sequence of nucleotides in DNA leaving free the simple chains that are ca led "sticky ends" and to which a foreign DNA can bind, as long as their bases are complementary

2) Incorporate DNA into a suitable mole.

cular vehicle. The DNA fragments obtained in the previous step cannot directly enter the cell of interest. To do so, each fragment must be incorporated into another DNA molecule called a vector, which essentially behaves as a molecular vehicle that can enter the host cell, transport foreign DNA and replicate it.

There are several types of vectors, but here we will refer specifically to plasmids that, as you will remember, are DNA rings indepen dent of the bacterial chromosome.

3) Recombine the DNA. To place the DNA fragment in the vector, both must be cut with the same restriction enzyme to generate "sticky ends" that can be joined by the complementarity of bases. Subsequently, both samples are mixed to facilitate the formation of hydrogen bonds between the bases and, with help of the enzyme DNA ligase, the sticky ends are permanently sealed by forming bonds between sugar and phosphates in the nucleotide chain. The DNA fragment integrated into the plasmid is an example of recombinant DNA obtained from the binding of DNA that comes from two different sources

4) Transform bacterial cells. The recombi nant plasmid obtained in step 3 is mixed with bacterial cells (usually with the so-called Escherichia coli) which are previously subject ted to chemical treatment or electric shocks in order to improve their ability to enter the DNA of the environment. Some of these bacteria capture the plasmid or recombinant DNA through the transformation process ex plained above

5) Clone the recombinant DNA. The bacte ria that captured the recombinant DNA are selected and maintained in a culture under special conditions to obtain a large popula tion. Each time the bacterium divides it will replicate the recombinant DNA and gene rate genetically identical daughter cells, called clones. Finally, these bacteria will be subjected to another process so that the gene expresses the protein or product of interest and it's possible to take advantage of it

Polimerase

1) The mixture contained in the test tube is heated to about 90-95 °C, which allows the hydrogen bonds that hold the two DNA strands together to break obtaining single stranded DNA.

2) The reaction temperature is lowered to 50-70 °C allowing primers to adhere to single stranded DNA.

3) In this step DNA polymerase is added, this recognizes the primers as sites to begin DNA synthesis. This synthesis is carried out at an approximate tempe rature of 70 °C. DNA polymerase adds free nucleotides to primers producing two new DNA strands.

Gel electrophoresis

Restriction enzymes allow DNA of interest to be cut into small pieces, resulting in a set of fragments of genetic material of different sizes. The separation of these segments allows to analyze the results of the polymerase chain reaction, study the structure of the proteins, investigate samples found in crime scenes or perform paternity tests. To achieve this, a technique called gel electrophoresis is used; in it the fragments are separated by size when applying an electric field.