Introduction to Cloning PDF

Introduction to Cloning Cloning is the process of producing genetically identical individuals of an organism either naturally or artificially.... Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or DNA fragments (molecular cloning). Cloning in biotech...

Introduction to Cloning Cloning is the process of producing genetically identical individuals of an organism either naturally or artificially.... Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or DNA fragments (molecular cloning). Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. Manipulation of DNA Gene manipulation is also sometimes called the genetic engineering. It is a general term for any method which manipulate with the genetic material. Gene manipulation includes gene splicing, use of recombinant DNA, forming of the monoclonal antibodies or PCR (polymerase chain reaction). The Process of the Gene Manipulation 1. To isolate DNA from the organism. 2. To put the DNA into the DNA vector. 3. To transfer the vector by transfection or transformation into the host. Isolation of the DNA We can get the part of the DNA thanks to restriction endonucleases - type II. The DNA strand is broken up in the specific places which are bordered by short nucleotide sequences. Both strands are cut out. Then we can recognize them and categorize by their size by agarose gel electrophoresis. Joining the DNA The DNA ligase governs the properties of the segment. The cohesive ends are created and the insertion can takes place. Transfer into the Host It is absolutelly necessary to choose the right vector for the DNA. It must accept the foreign DNA and continues its cell cycle. The most common are bacteria – especially E.coli. The transformation of DNA from a virus is called transfection. We have ensured that the integration into the host genome will be successful. The most important is to maintain the ability to replicate DNA. Vector in molecular biology means a molecule of DNA, which serves as a carrier of genetic information into the cells. It is used especially in molecular genetic. Generally it is consisting of inserted DNA sequence and larger DNA sequence which serves as a supporting structure. The most common vectors are plasmids, viruses and artificial chromosomes. Vectors are used especially to transfer of genetic information into the cells in order to replication and expression of the selected part of DNA. Whole process is induced by promoter which is also contained in vector DNA. Inserting vector into cells is called due to target cell: transformation (for bacterial cells), transfection (for eukaryote), transduction (for viruses). Plasmids Plasmids are molecules with double-stranded circular DNA structure. Their transfer into cell is connected with translation into mRNA and transcription into proteins. They carry the inserted gene and the necessary enzymes and control molecules. Most often they are applied to bacteria that are cultivated on soils for hundreds and thousands of copies. Vectors can then be removed from the bacteria using restriction enzymes and get the individual parts of DNA. The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Plasmids used as vectors maintain a modified origin of replication that allows their replication within the host, and they contain a gene for antibiotic resistance which ensures that, following treatment with a high dose of antibiotic, all viable bacterial colonies will contain several copies of the plasmid. an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription factors. a promoter is a region of DNA that leads to initiation of transcription of a particular gene. In genetics, a transcription terminator is a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription Transcription Initiation Site. The first nucleotide of a transcribed DNA sequence where RNA polymerase (DNA-DIRECTED RNA POLYMERASE) begins synthesizing the RNA transcript. Translation initiation is a key step for regulating the synthesis of several proteins. In bacteria, translation initiation involves the interaction of the mRNA with the ribosomal small subunit. Viral vectors Viral vectors are often artificially constructed and contain modified DNA or RNA. Their treatment is shedding its own infectivity but retain the ability to penetrate and propagate in the cell. It infectivity deprivation can sometimes lead to the need for co-operation with another virus. It is necessary for successfully transfection. Viral vectors are often incorporates into the host genome. Most common are retroviruses.