From the double helix to the genome

The computer proved an indispensable tool in managing, Bioinformatics Genetic engineering has boosted the development of different branches of science such as biochemistry, genetics and molecular biology. The computer proved an indispensable tool in managing, processing and connecting the huge amount of data that emerged from genetic techniques. BIOINFORMATICS From the double helix to the genome> Bioinformatics

modify the expression of one or more genes. Recombinant DNA Bioengineering techniques for the manipulation and analysis of DNA outside the cell. recombinant DNA technology makes it possible to isolate and cut short sequences of DNA before transferring and inserting them into the genome of other cells in order to modify the expression of one or more genes. Targeted transfer of DNA may also take place between the genetic material belonging to different species. From the double helix to the genome > Recombinant DNA

Cloning In 1973, Stanley Cohen and Herbert Boyer began to develop a new technique called cloning and managed to create the first “genetically modified” organism in history. From the double helix to the genome > Cloning

Technique of cloning It is possible: to insert the gene region of interest into a plasmid to produce large quantities of the cloned region through the transfer of the vector into bacteria to select only the bacteria that contain the recombinant vector a gene for resistance to an antibiotic is used. From the double helix to the genome >Technique of cloning

Enzymes and restriction sites Restriction enzymes are proteins capable of cutting the DNA at precise nucleotide sequences, called restriction sites. Restriction enzymes are produced by bacteria to protect themselves from viruses that parasitize them, cutting their DNA. are able to act on the foreign DNA only. From the double helix to the genome > Enzymes and restriction sites

specific palindrome sequences, forming sticky ends. A restriction site is a palindromic sequence of 4, 6 or 8 pairs of nitrogenous bases. The majority of restriction enzymes work by making a staggered cut at the level of their specific palindrome sequences, forming sticky ends. From the double helix to the genome > Enzymes and restriction sites

PCR: the polymerase chain reaction Starting in 1985, this technique was designed allowing the in vitro amplification of DNA. PCR is a technique that, using a DNA polymerase, allows the rapid production of millions of identical copies of a DNA region, starting from extremely small quantities of material. The nucleotide sequences that surround it perfectly. Is the ‘moulds’ for the in vitro synthesis of the triggers from which the duplication reaction of the target region will then take place. The proteins and nucleic acids > PCR: the polymerase chain reaction

The primers The primers, that initiate the reaction, are two sequences of 17-30 nucleotides, complementary to the sequences of the target DNA that constitute, respectively, the beginning of a filament and the end of it on the opposite strand. The proteins and nucleic acids > The primers

The thermal cycler The replication reaction of the DNA region of interest takes place in a machine called a “thermal cycler” and regulates the succession of amplification cycles during which it alternates 3 different temperatures: 94 ˚C: denaturation of double-stranded DNA template into two single strands through heating; 30-65 ˚C: annealing of the primers to the sequences of single-stranded DNA 65-75 ˚C: primer extension by addition of deoxyribonucleotides in the 5’→3’ direction carried out by the DNA polymerase. The proteins and nucleic acids > The thermal cycler

Gene sequencing The genome as the ‘instruction manual’ that describes the functioning of all living organisms. MAP OF THE GENES PHYSICAL MAPPING GENETIC MAPPING the relative distance between genes based on the techniques used in molecular biology to build maps that show the physical location of the genes The proteins and nucleic acids > Gene sequencing

only one –OH group has been replaced The Sanger method The turning point for DNA sequencing was determined by the possibility of using DNA polymerase outside the cellular environment. 1975, The Sanger method. A terminator is a molecule of dideoxyribonucleotide triphosphate (dNTP), that is to say a molecule of deoxyribonucleotide triphosphate (dNTP) in which, in the molecule of ribose, only one –OH group has been replaced with a hydrogen atom. The proteins and nucleic acids > DNA sequencing with terminators

behaviours under the action of an electric field. Electrophoresis of DNA fragments The Sanger method of DNA sequencing provides a mixture of DNA fragments which must be separated The Gel Electrophoresis Technique Gel electrophoresis is a method that exploits the chemical characteristics of molecules (nucleic acids or proteins) to separate them and identify them thanks to their differing behaviours under the action of an electric field. The proteins and nucleic acids > Electrophoresis of DNA fragments

The identification of the function of each gene An incomplete genome: Dark DNA The Human Genome Project The goal (1987) was to determine the nucleotide sequence of human genetic makeup, identifying and mapping the genes that make it up - a goal that was achieved in 2003. HOWEVER The identification of the function of each gene was still a long way off. The proteins and nucleic acids > An incomplete genome: Dark DNA

Outcomes and evidences Only 2% of the human genome consists of genes and the remaining part which normally does not encode, was hastily termed “junk DNA”. DNA is not the sole repository of genetic information. Non-coding DNA may still play a role in gene expression, backup or in regulation. It serve to ‘switch’ genes ‘on’ or ‘off’, thus triggering, or blocking. At the end of the project, however, they had identified ‘only’ 20- 30,000 genes, instead of the 100,000 expected. The complexity of an organism does not only depend on the number of genes contained in its DNA. The proteins and nucleic acids > Outcomes and evidences

The Encode project Deeper study of the human genome and to obtain a map, which identifies 4 million new stretches of DNA that, in effect, act as (on-off) switches in the regulation of gene expression. The proteins and nucleic acids > The Encode project