Biotechnology
Biotechnologies: techniques and applications
Molecular cloning is a technology used to obtain several copies of DNA strands, introducing them into host organisms with the use of molecular vectors.
DNA molecules obtained by molecular cloning are called recombinant DNA.
The first step of molecular cloning is the isolation of a DNA sequence. It is possible to clone a DNA strand or the DNA copy of an mRNA; the latter is the most common approach in biotechnologies. The DNA copy of an mRNA is called cDNA (complementary DNA), which can be combined into a cDNA library.
Reverse transcriptase is the enzyme that generates cDNA from an mRNA template. It first copies the single strand mRNA, producing a double strand DNA/RNA hybrid.
Reverse transcriptase can degradate the RNA strand, leaving a single strand DNA. It then synthesizes the complementary DNA strand, generating a double strand cDNA.
Circular DNA molecules (plasmid vectors) can be used to introduce cDNA into host cells. Plasmids are genetic elements found in bacteria that can be modified in lab.
Plasmid vectors have an origin of replication (ori), a genetic marker (e.g. antibiotic resistance) and restriction sites that can be recognized by restriction enzymes.
Restriction endonucleases are enzymes that can recognize specific DNA sequences (restriction cleavage sites) and cut the double helix. The activity of DNA ligase is necessary for the cDNA to be inserted into a vector. This enzyme catalyzes the formation of a phosphodiester bond between two adjacent nucleotides, joining two DNA strands together. The cDNA sequences and the cloning vector are treated with
the same restriction endonuclease so that the ends of the vector and the cDNA are complementary. DNA ligase joins together cDNA inside plasmid vectors.
Recombinant DNA is inserted into bacterial cells, through a process called
transformation. Only some cells receive plasmid DNA. Cells containing the vector have gained antibiotic resistance; it is therefore possible to select them by putting the cells in a culture medium that contains the antibiotic. The polymerase chain reaction (PCR) is a technology used to obtain several copies of a DNA sequence. It consists of a series of cycles of denaturation, annealing and elongation. At the end, the starting sequence is amplified at every cycle n of a factor 2n (32 cycles = 232 copies = >109 copies). In order to “read” the type and order of nucleotides in a DNA molecule, it is necessary to have:
• DNA template strand – the sequence we want to know;
• a primer complementary to a region in the DNA template;
• DNA polymerase;
• mix of 4 dNTPs (deoxynucleotides);
• fluorescently labeled ddNTPS (dideoxynucleotides).
DNA polymerase is incubated with a DNA template, as well as 4 dNTPs and one specific ddNTP which stop the chain elongation. With 4 separate reactions, one
for each ddNTP, it is possible to identify every base in the DNA template.
PCR technology allows the use of small amounts of DNA to obtain a sequencing.
In 2003 the Human Genome Project announced the complete sequencing of the human genome. It was a publicly funded, international research project that began in 1990.
The results of the project are useful in many fields:
• identifying mutations associated with different diseases;
• designing more efficient drugs and treatments;
• studying human evolution;
• improving forensic sciences.
Biotechnology changed the scientific research in criminal investigations:
• with the PCR it is possible to amplify even tiny amounts of DNA from biological samples;
• it is possible to provide a unique genetic profile from a sample of DNA.
DNA fingerprinting is the technique used to characterize the unique profile that can distinguish between two individuals, based on a DNA sequence.
The technique uses highly variable and repetitive short sequences (short tandem repeats, STR), made up of repetitions of 3-5 nucleotides. After amplification with PCR, the products are displayed on agarose gel.
Biotechnologies can be used to modify the genome of some organisms (GMO, genetically modified organisms), in order to generate new varieties or to cancel, induce or change a characteristic of the organism. It is possible to generate genetically modified animals or plants. In agriculture, some species are subjet to genetic modification in order to obtain:
• better ripening processes;
• resistance to herbicide (or other chemical treatments);
• resistance to stress (cold, salinity, drought);
• resistance to pests (viruses, bacteria, insects, fungi);
• better nutritional value;
• production of exogenous recombinant proteins (enzymes, antibodies, antigenes for vaccines).
Resistance to herbicides: corn, cotton and soy are transfected with genes that allows crops to resist the use of herbicides.
Resistance to stress: transected with the MAP kinase gene from the plant Arabidopsis thaliana confers resistance to cold, heat and salinity.
Resistance to pests: tobacco and zucchini transfected with a gene from the tobacco mosaic virus; cotton and corn transfected with the Bt toxin from Bacillus thuringiensis.
Biotechnologies: techniques and applications
Molecular cloning is a technology used to obtain several copies of DNA strands, introducing them into host organisms with the use of molecular vectors.
DNA molecules obtained by molecular cloning are called recombinant DNA.
The first step of molecular cloning is the isolation of a DNA sequence. It is possible to clone a DNA strand or the DNA copy of an mRNA; the latter is the most common approach in biotechnologies. The DNA copy of an mRNA is called cDNA (complementary DNA), which can be combined into a cDNA library.
Reverse transcriptase is the enzyme that generates cDNA from an mRNA template. It first copies the single strand mRNA, producing a double strand DNA/RNA hybrid.
Reverse transcriptase can degradate the RNA strand, leaving a single strand DNA. It then synthesizes the complementary DNA strand, generating a double strand cDNA.
Circular DNA molecules (plasmid vectors) can be used to introduce cDNA into host cells. Plasmids are genetic elements found in bacteria that can be modified in lab.
Plasmid vectors have an origin of replication (ori), a genetic marker (e.g. antibiotic resistance) and restriction sites that can be recognized by restriction enzymes.
Restriction endonucleases are enzymes that can recognize specific DNA sequences (restriction cleavage sites) and cut the double helix. The activity of DNA ligase is necessary for the cDNA to be inserted into a vector. This enzyme catalyzes the formation of a phosphodiester bond between two adjacent nucleotides, joining two DNA strands together. The cDNA sequences and the cloning vector are treated with
the same restriction endonuclease so that the ends of the vector and the cDNA are complementary. DNA ligase joins together cDNA inside plasmid vectors.
Recombinant DNA is inserted into bacterial cells, through a process called
transformation. Only some cells receive plasmid DNA. Cells containing the vector have gained antibiotic resistance; it is therefore possible to select them by putting the cells in a culture medium that contains the antibiotic. The polymerase chain reaction (PCR) is a technology used to obtain several copies of a DNA sequence. It consists of a series of cycles of denaturation, annealing and elongation. At the end, the starting sequence is amplified at every cycle n of a factor 2n (32 cycles = 232 copies = >109 copies). In order to “read” the type and order of nucleotides in a DNA molecule, it is necessary to have:
• DNA template strand – the sequence we want to know;
• a primer complementary to a region in the DNA template;
• DNA polymerase;
• mix of 4 dNTPs (deoxynucleotides);
• fluorescently labeled ddNTPS (dideoxynucleotides).
DNA polymerase is incubated with a DNA template, as well as 4 dNTPs and one specific ddNTP which stop the chain elongation. With 4 separate reactions, one
for each ddNTP, it is possible to identify every base in the DNA template.
PCR technology allows the use of small amounts of DNA to obtain a sequencing.
In 2003 the Human Genome Project announced the complete sequencing of the human genome. It was a publicly funded, international research project that began in 1990.
The results of the project are useful in many fields:
• identifying mutations associated with different diseases;
• designing more efficient drugs and treatments;
• studying human evolution;
• improving forensic sciences.
Biotechnology changed the scientific research in criminal investigations:
• with the PCR it is possible to amplify even tiny amounts of DNA from biological samples;
• it is possible to provide a unique genetic profile from a sample of DNA.
DNA fingerprinting is the technique used to characterize the unique profile that can distinguish between two individuals, based on a DNA sequence.
The technique uses highly variable and repetitive short sequences (short tandem repeats, STR), made up of repetitions of 3-5 nucleotides. After amplification with PCR, the products are displayed on agarose gel.
Biotechnologies can be used to modify the genome of some organisms (GMO, genetically modified organisms), in order to generate new varieties or to cancel, induce or change a characteristic of the organism. It is possible to generate genetically modified animals or plants. In agriculture, some species are subjet to genetic modification in order to obtain:
• better ripening processes;
• resistance to herbicide (or other chemical treatments);
• resistance to stress (cold, salinity, drought);
• resistance to pests (viruses, bacteria, insects, fungi);
• better nutritional value;
• production of exogenous recombinant proteins (enzymes, antibodies, antigenes for vaccines).
Resistance to herbicides: corn, cotton and soy are transfected with genes that allows crops to resist the use of herbicides.
Resistance to stress: transected with the MAP kinase gene from the plant Arabidopsis thaliana confers resistance to cold, heat and salinity.
Resistance to pests: tobacco and zucchini transfected with a gene from the tobacco mosaic virus; cotton and corn transfected with the Bt toxin from Bacillus thuringiensis.
