Expand PCR. How it helps in amplification of gene of interest ?

Kary Mullis invented the technique known as PCR (Polymerase chain reaction) in the year 1983. The polymerase chain reaction is a process in which many copies of the DNA sequence are made. The denaturation is a principle of PCR. The PCR is an essential process in biotechnology because, in every living organism, DNA is unique. It is a widespread technique used for making millions of DNA sequences. The polymerase chain reaction is an important technique used to make multiple copies of DNA sequence or the desired gene in the laboratory. Amplification of gene is done by using three different steps, which are as follows: Denaturation: When high temperature is applied for15 seconds, the two strands separate to start the process of amplification. The step occurs at a temperature of 95ºC Annealing: The primer in two set are added at the two ends of 3ʹ site. This step occurs at a temperature of 55ºC. Extension: The DNA polymerase uses a thermostable Taq polymerase, which causes the polymerization of the template strand by adding the nucleotides. These three steps repeat many times which help in obtaining many copies of DNA.

Explain any two methods for introduction of recombinant DNA. into host cell.

The recombinant DNA (deoxyribose nucleic acid) technology involves the cleaving of DNA at a particular site (using molecular scissors), and inserting them in the virus, cloning vectors or plasmids. This is known as genetic engineering or recombinant DNA technology. The two methods of insertion of foreign DNA into the host can be achieved by electroporation and microinjection. Electroporation:-DNA is a hydrophilic molecule so, it cannot pass through cell membranes. In turn, to force bacteria to take up the plasmid, the bacterial cells must be made competent to take up DNA. This is completed by treating them with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can be then forced into such cells by incubating the cells with recombinant DNA on ice followed by placing them briefly at 42⁰C and then putting them back on ice. This enables the bacteria to take up the recombinant DNA. Microinjection – The technique of transferring the gene of interest directly into the host cell by using the microinjection. The microinjection is a glass micropipette consisting of an extremely thin needle of diameter 0.1 microns. This thin needle is used to directly inject the recombinant DNA into the nuclei of the host cell. This process does not involve any additional pressure to transfer the desired gene. Although it requires extensive practice and is expensive equipment in which only a few numbers of cells can be used to create the recombinants. For example, this technique is widely used to create a “giant mice” by injecting the gene responsible for growth hormone in rats into the mice.

What is the role of Ori for cloning vector?

The cloning vector can be taken from DNA or a virus it can be the cell of a higher organism or the plasmid of a bacterium. Ori or Origin of replication is the site where the DNA replication starts. When any DNA fragment is attached to it, and the replication starts within the cell. Ori is necessary for the cloning vector to produce multiple numbers of copies. It also checks the number of copies generated. A phagemid is a DNA based cloning vector, which has both bacteriophage and plasmid properties. This vector carries the origin of plasmid replication.Multiplication of the genetic material between generations requires accurate duplication of DNA by semiconservative replication before cell division to confirm each daughter cell receives the full complement of chromosomes.