Recombinant DNA (rDNA)
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
Recombinant DNA was first achieved in 1973 Herbert Boyer, of the University of California at San Francisco, and Stanley Cohen, at Stanford University, who used E. coli restriction enzymes to insert foreign DNA into plasmids. [12]
Recombinant DNA is the general name for a piece of DNA that has been created by the combination of at least two strands. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the nucleotide sequence within that identical overall structure.
Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.
The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA.
In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules.
Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms.
Proteins that can result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced.[13]
Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring by foreign coding sequences.[14]
Recombinant DNA differs from genetic recombination in that the former results from artificial methods in the test tube, while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms.
The Basics of Recombinant DNA
That's a very good question! rDNA stands for recombinant DNA. Before we get to the "r" part, we need to understand DNA.Those of you with a background in biology probably know about DNA, but a lot of ChemE's haven't seen DNA since high school biology. DNA is the keeper of the all the information needed to recreate an organism. All DNA is made up of a base consisting of sugar, phosphate and one nitrogen base. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen bases are found in pairs, with A & T and G & C paired together.
The sequence of the nitrogen bases can be arranged in an infinite ways, and their structure is known as the famous "double helix" which is shown in the image below. The sugar used inDNA is deoxyribose. The four nitrogen bases are the same for all organisms. The sequence and number of bases is what creates diversity. DNA does not actually make the organism, it only makes proteins. The DNA is transcribed into mRNA and mRNA is translated into protein, and the protein then forms the organism. By changing the DNA sequence, the way in which the protein is formed changes.
transcribe 기록하다
This leads to either a different protein, or an inactive protein. Now that we know what DNA is, this is where the recombinant comes in. Recombinant DNA is the general name for taking a piece of one DNA, and combining it with another strand of DNA. Thus, the name recombinant! Recombinant DNA is also sometimes referred to as "chimera." By combining two or more different strands of DNA, scientists are able to create a new strand of DNA.The most common recombinant process involves combining the DNA of two different organisms.
How is Recombinant DNA made?
There are three different methods by which Recombinant DNA is made. They areTransformation, Phage Introduction, and Non-Bacterial Transformation. Eachare described separately below.
Transformation
The first step in transformation is to select a piece of DNA to be inserted into a vector. The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules.
An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant. The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance, colorchanges, or any other characteristic which can distinguish transformed hosts from untransformed hosts. Different vectors have different properties to make them suitable to different applications. Some properties can include symmetrical cloning sites, size, and high copy number.
application 지원
Non-Bacterial Transformation
This is a process very similar to Transformation, which was described above. The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.
Phage Introduction
Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packaging of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.
Working of rDNA
Recombinant DNA works when the host cell expresses protein from the recombinant genes. A significant amount of recombinant protein will not be produced by the host unless expression factors are added.
Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the transcription and translation of the gene by the cell.
transcription and translation 전사와 번역
These signals include the promoter, the ribosome bindingsite, and the terminator. Expression vectors, in which the foreign DNA is inserted,contain these signals.
Signals are species specific. In the case of E. Coli, these signals must be E. Coli signals as E.Coli is unlikely to understand the signals of human promoters and terminators. Problems are encountered if the gene contains introns or contains signals which act as terminators to a bacterial host.
This results in premature termination, and the recombinant protein may not be processed correctly, be folded correctly, or may even be degraded. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi.
premature 너무 이른
termination 종료
degrade 분해되다
The use of animal cells is difficult due to the fact that many need a solid support surface, unlike bacteria, and have complex growth needs. However, some proteins are too complex to be produced in bacterium,so eukaryoticcells must be used.
Importance rDNA
Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only become more important in the 21st century as geneticdiseases become more prevelant and agricultural area is reduced. Below aresome of the areas where Recombinant DNA will have an impact.
Better Crops (drought & heat resistance)
Recombinant Vaccines (Hepatitis B)
Prevention and cure of sickle cell anemia
Prevention and cure of cystic fibrosis
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy [15]