Tools of Recombinant DNA technology
The tools mainly include the following:
1. The enzymes which include the restriction enzymes – help to cut, the polymerases- help to synthesize and the ligases- help to bind.
synthesize 합성하다
The restriction enzymes used in recombinant DNA technology play a major role in determining the location at which the desired gene is inserted into the vector genome.
They are two types, namely Endonucleases and Exonucleases. The Endonucleases cut within the DNA strand whereas the Exonucleases remove the nucleotides from the ends of the strands.
The restriction endonucleases are sequence-specific which are usually palindrome sequences and cut the DNA at specific points. They scrutinize the length of DNA and make the cut at the specific site called the restriction site.
restriction 제한
This gives rise to sticky ends in the sequence. The desired genes and the vectors are cut by the same restriction enzymes to obtain the complementary sticky notes, thus making the work of the ligases easy to bind the desired gene to the vector.
2. The vectors – help in carrying and integrating the desired gene. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism.Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have very high copy number.
The vectors are made up of an origin of replication- This is a sequence of nucleotide from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted
3. Host organism – into which the recombinant DNA is introduced. The host is the ultimate tool of recombinant DNA technology which takes in the vector engineered with the desired DNA with the help of the enzymes.There are a number of ways in which these recombinant DNAs are inserted into the host, namely – microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc.
Recombinant DNA Technology (With Diagram)
Steps in Recombinant DNA Technology[17] :
i. Selection and isolation of DNA insert
ii. Selection of suitable cloning vector
iii. Introduction of DNA-insert into vector to form rec DNA molecule
iv. rec DNA molecule is introduced into a suitable host.
v. Selection of transformed host cells.
vi. Expression and multiplication of DNA-insert in the host.
(i) Selection and isolation of DNA insert:
First step in rec DNA technology is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.
(ii) Selection of suitable cloning vector:
A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rec DNA technology. Most commonly used vectors are plasmids and bacteriophages.
integrate 통합시키다
(iii) Introduction of DNA-insert into vector to form recDNA molecule:
The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes [in step (i)] are now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA molecule which is often called as cloning-vector-insert DNA construct.
extract 추출하다
cleave 쪼개다
ligate 묶다
(iv) rec DNA molecule is introduced into a suitable host:
Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)] is introduced into these host cells. This process of entry of rec DNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however yeast, fungi may also be utilized.
(v) Selection of transformed host cells:
Transformed cells (or recombinant cells) are those host cells which have taken up the recDNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.
(vi) Expression and Multiplication of DNA insert in the host:
Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.