Tuesday, May 2, 2017

Genetics (8/8): DNA and genomic technology

Most of the methods used to clone DNA fragments have some common features, for example there is a method that uses bacteria and their plasmids.
Plasmids are small molecules of circular DNA that replicate within bacterial cells, regardless of the bacterial chromosome.
In order to clone genes or fragments of DNA isolate the bacterial plasmids, insert an extraneous gene inside the plasmid and finally reenter it into the bacterial cell where it is reproduced by forming a cell clone that also contains the foreign gene and this bacterial clone It will produce the protein encoded by the foreign gene.
Cloning can be used to obtain protein products for research or mass production of specific genes.

Restriction enzymes are enzymes that cut DNA molecules on a limited number of specific regions.
Restriction enzymes protect bacteria from foreign DNA and work by cutting this DNA through a process known as restriction.
Most of these enzymes are highly specific, and the bacterial cell protects its DNA from restriction by the addition of methyl groups (-CH3) within sequences recognized by restriction enzymes.
The recognition sequence is referred to as a restriction site, these sites are usually symmetrical and have the same sequence 5 '-> 3' consisting of 4 or 8 nucleotides, recognizable on both filaments but oriented in opposite directions.
Restriction enzymes cut the bonds on both filaments, and since these sequences are usually present in the DNA multiple times, the same enzyme can perform more cuts.
When subjected to the action of a given enzyme, copies of a DNA molecule always generate the same set of restriction fragments, so a restriction enzyme cuts a DNA molecule in a reproducible way.
In the product fragments there is at least a short single strand end region, said adhesive end, which will temporarily collide with few hydrogen bonds on the single strand of the other DNA molecules cut with the same enzyme, and these couplings can be stabilized with the enzyme DNA ligase, which holds the filaments together, catalyzing the formation of phosphodiester bonds.
It has recombinant DNA, that is, a molecule obtained from the union of DNA from two different sources.

The original plasmid is called a cloning vector and is a DNA molecule that can carry foreign DNA inside the cell and replicate it.

The cloning in a bacterial plasmid
There are 5 steps for cloning a gene:

  1. Isolation of the vector and DNA of the gene to be cloned
  2. Insertion of DNA into the vector: By the restriction enzyme, foreign DNA and plasmid DNA are cut off and the various fragments will melt together thanks to the adhesive ends, which will then be ligated by DNA ligase with covalent bonds.
  3. Introducing the cloning vector into the cells: in some cases through the process of transformation.
  4. Cell cloning
  5. Identification of clones: hybridization can be used following the probe labeled with radioactive isotopes that will bind to the filaments of the desired gene, then use DNA denaturation to separate the 2 filaments.

Cloning for eukaryotes in prokaryotes
An expression vector is a cloning vector that contains the procariotic promoter necessary to obtain a cloned eukaryotic gene that functions in a prokaryotic system.
Complementary DNA (cDNA) is that DNA that is produced by reverse transcriptase of eukaryotic mRNA to subclude the incompatibility of eukaryotic DNA (full of introns) with that of procariotic target.
Artificial yeast chromosomes (YACs) are vectors that combine the essential characteristics of the eukaryotic chromosome with foreign DNA.
Electroporation occurs when an electrical impulse is applied to the cell-containing solution by which a hole is formed in the cytoplasmic membrane from which DNA enters the cell.
Eucariotic cloned gene expression is used because eukaryotic cells because many proteins are not modified after translation do not work, and prokaryotes can not modify them.

The genomic library is the set of recombined plasmids, each of which contains a particular segment of the initial genome (there is also the cDNA library).

Polymerase Chain Reaction (PCR) is a technique by which any piece of DNA can be quickly copied several times without the use of cells.
The DNA is incubated in a test tube in the presence of polymerase, nucleotides and short filaments of single-stranded synthetic DNA, and PCR thus allows to generate billions of copies of a specific segment of DNA in a few hours in a 3-cycle cycle , Where there is no need to start from a pure sample, but a small amount of DNA involved, however, PCR commits several occasional errors and therefore can not replace cloning when it takes many copies.


DNA and genomic analysis


Genomics are concerned with the analysis of whole genome sequences so that they can be used as a starting point for the study of various gene sets and their interactions.
Gel electrophoresis is a technique that allows the separation of macromolecules based on their size, electrical charge or other physical properties, it separates the macromolecules based on their migration speed on a gel placed within a field electric.
The electrophoresis divides a mixture of DNA molecules into bands, each of which is made up of DNA moleocles of the same length.

Restriction fragment analysis indirectly detects sequence differences between DNA molecules by electrophoresis on gel.
Many molecules can be identified by observing their frameworks of restriction fragments, which can be retrieved in order to obtain pure samples.

The southern blotting technique can be used to compare the DNA of different subjects.
This technique is based on the nucleic acid hybridization, and the results may show both the presence of a particular sequence in a DNA sample, but also the restriction fragments that contain that sequence.
First, restriction fragments (DNA + restriction enzyme) are prepared, then the mixture of restriction fragments of each sample is separated by electrophoresis, then blotting is used, by means of an alkaline solution the individual DNA strands remain attached to the paper, Banded, there is hybridization with the radioactive probe and finally autoradiography allows to detect the DNA bands that appear with the probe.

Restriction fragment length polymorphisms (RFLPs) are differences in DNA sequences of homologous chromosomes, which give rise to different types of restriction fragments.
The RFLP can be used as a genetic marker of a particular locus of the genome.
RFLPs are identified and studied by southern blotting, and since RFLPs are Mendelian inherited, they can be used as genetic markers to construct association maps, and the frequency with which two RFLPs are inherited together is a measure of proximity Of the 2 loci on a chromosome.

In 1980, biologist David Botstein stated that DNA variations observed in RFLP could be used as a basis for detailed mapping of the human genome.
In 1990, however, the human genome project began to map the entire human genome through the determination of the complete nucleotide sequence of DNA of each chromosome, a project developed in 3 phases:
  1. Genetic Mapping: The construction of a map of association of the many thousands of genetic markers present in the chromosomes, and relying only on the microsatellite, the researchers completed a human genetic mapping with about 5000 markers.
  2. Physical mapping: The distances between the markers are expressed by a physical measure (usually with the number of nucleotides), and the DNA of each chromosome is cut into a number of identifiable restriction fragments (cloned) by determining the actual order Fragments in the chromosome.
    The overlapping fragments are identified by probes using the chrmosome walking method.
    The scientists determine the order of the long fragments and then cut each of them into smaller pieces that are cloned and sorted.
  3. DNA Sequencing: It is the complete nucleotide sequence of a genome that starts from the ordered DNA fragments, whose nucleotide sequence can be determined by the use of a sequencer.
    The rapid sequencing technique marks the DNA and is synthesized by the use of special nucleotides ending the chain and by the heprophoresis on high resolution gel.

In 1992, biologist Craig Venter proposed an alternative approach to the sequencing of whole genomes, proposing to skip the first two mapping phases and to go directly to sequencing through the use of powerful computers.
The human genome is in the process of being completed, but some interesting things have been discovered, such as the fact that the human genome is 85% equal to that of the mouse.

To facilitate research, the DNA sequences already analyzed are collected in electronic databases reachable by researchers around the world.
Among the various discoveries made, there is one that the human genome contains only a few genes, about 30000-40000, only 2-3 times higher than those of the fruit fly, and that solved a very small portion of human DNA is represented by genes , The remainder consists of repetitive DNA and long introns.
In addition, the comparison of genomic sequences fully confirms evolutionary links even among very distant organisms.
A typical human gene usually has at least 2 or 3 different polypeptides, using different combinations of exons.

Study of gene expression
Genome study is important for both knowing new genes and understanding how they evolve, and to understand how genes act together to create and function an organism.
By using the DNA microarray technique, small amounts of numerous single-stranded DNA fragments representing distinct genes are fixed on a slide and hybridized with different dye-labeled cDNA samples.
This technique serves to identify new genes, understand how they interact and how they work, and this technique is also used to compare cancerous tissues with healthy tissues.

Determine the functioning of the genes
To understand how a gene works, sometimes it disables and looks at differences in the body, a method is that of in vitro mutagenesis, a technique that introduces specific changes in the sequence of a cloned gene, mutations that can block the function of Protein product when it is reinserted into the cell.
With RNA interference (RNAi), genes expression is stopped by using double-stranded synthetic RNA molecules that correspond to the sequence of a particular gene to trigger the destruction of the gene-matching messenger RNA.

Proteomics is the systematic study of the whole set of proteins encoded by the genome.
The number of proins is much higher than that of genes, due to alternative splicing and post-translational modifications.
Bioinformatics is the application of computer science and mathematics to genetics and other fields of biology.
Single-nucleotide polymorphisms (SNPs) are variations of single genome-based pairs that are the cause of our genomic diversity, small diversity compared to other species.


Practical applications of DNA technology


Scientists can diagnose hundreds of human genetic disorders by engaging DNA technology through human genome study to identify disease mutations.

Gene therapy is the alteration of the genes of an individual suffering from some disease.
Cells should receive a normal allele and duplicate, however, this method has for now little success because of the brevity of the functioning of these genes.

Thanks to DNA technology, various pharmaceutical products, mainly protein, have been created, for example, insulin and growth hormone (hGH) and the tissue plasminogen activator (TPA) have been produced that helps prevent the heart attack.
The flaw of these products is that they are very expensive.
Another example of genetic product is the vaccine is an innocuous variant or a derivative of a pathogen that stimulates the immune system to fight the pathogen.
The vaccines are of 2 types: inactivated virulent viral particles and attenuated viral strain active virus particles.

DNA testing is also used in the legal field as it may lead to the identification of a criminal offense, through RFLP analysis by Southern blotting that requires fewer amounts of blood or other tissue.
DNA fingerprint, DNA fingerprint, is a specific profile of bands that can be used in the legal field, and it is recently produced using variations in the length of the satellite DNA instead of RFLPs, since these simple tandem repeats (STR ) Vary from person to person and are considered reliable.
The higher the number of DNA markers tested in a sample and the more the fingerprint of an individual is unique.
PCR is useful when DNA is of low quality or in small quantities.
Legal DNA examinations focus only on 5 small genome regions, which are known to have high variability among people, so that the possibility of having the same fingerprint is 1/100000 and 1 / a billion, depending on the Markers compared to the frequency of these markers within the population.

Another use is the environmental one, where, for example, some bacterial strains have been developed that can degrade the compounds released during oil spills at sea.

DNA technology is also used in agriculture to produce more resistant bacteria and insects, and produce more protein / vitamins.
Transgenic organisms are those organisms whose genes contain genes of other species, for example a sheep that produces better wool can be produced or a generic transgenic animal can be used as a drug factory.
The transgenic animal is generated by picking the egg cells from the receiving animal, cloning the gene from the donor animal, and implanting the egg with the gene into the recipient, which will make a transgenic offspring.
Plants are used because plant cells are easily manipulated and because it would be less expensive to produce human proteins and vaccines through plants than through classical cloning, and this is made possible because some plants are able to generate completely from a single cell.
The vector used to introduce new genes into plant cells is the plasmid Ti.

Because of various ethical issues, scientists have developed several self-control rules to prevent accidents with mutant genes that could be very dangerous by creating laboratories with very advanced security systems and generating experimental genes that can not live out of the lab.
On genetically modified organisms (organisms that have acquired one or more artificial genes) a lot has been discussed to decide whether it is ethically correct to alter nature, but its utility in fields such as agriculture or the food field is undisputed ( Animals), and some countries have decided to adopt a trademark to indicate GMOs when they are sold.

To avoid the spread of manipulated genes, researchers are also able to create them in a way that can not be transmitted, and to avoid the various risks of genetic technology, the United States has set up various control bodies, such as the Food and Drug Administration, National Institute of Health and the Department of Agriculture.
DNA technology is an important discovery, but it must be carried out with great care and caution.

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