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The whole genome sequencing to identify Mendelian disorders
Posted on June 9th, 2010 No commentsThe current way to determine the cause of disease is finding mutations via DNA sequencing. In order to reduce costs only coding regions are sequenced and analyzed. Unfortunately, several Mendelian traits that can be the basis for specific diseases are not present in coding regions.
Therefore, the sequencing of the whole genome might contribute better understand the causal variant of diseases. Scientists from the Institute for Systems Biology in Seattle proposed this approach to study the Mendelian hesitance of two recessive disorders. They analyzed the whole genomes of healthy parents and sick children. They delineated an accurate recombination map showing exactly which pieces of parental chromosomes had been assembled in offspring genetic material. Then, they corrected 70% of sequencing errors and especially they reduced the search space for the disease- causing variants. This study is important because it demonstrated that is possible to identify the genes involved in etiology of certain disease by sequencing the DNA of the family in which this disease appears. Based on this observation, scientists plan to analyze the genome of family with Huntington’s disease. This approach requires the absolute precision of sequencing data. -
The synthetic genome
Posted on May 28th, 2010 No commentsThis is the news of the day: scientists fro the Craig Venter Institute generated the first genome of a viable cells. The research has been published on Science Express and in few hours has been diffused and commented around the world. What is the story of this clamorous paper? Scientists used one yeast strain as a model. Firstly, they in silico designed the genome and placed some sequences of control in order to surely distinguish synthetic genome from the natural one. Previous experiences in sequencing helped researchers to well design the synthetic genome. Indeed, we have to remember that the Craig Venter Institute was one of the first institutions to complete genome sequencing of several organisms.
They spitted the synthetic genome into small portions and sequentially assembled them. They started from 1kbp units that were amplified in E. Coli, purified and transformed in yeast. Ten 1kbp formed the first 10kb intermediates. In order to control the intermediates quality, multiplex PCR was carried out, by using specific primers that bound the connecting sequences. They repeated the same process also to produce 100kb intermediates, but they directly amplified the DNA in yeast because E. Coli wasn’t able to do it. The final assembling required additional vector sequences and was performed in yeast spheroplasts. The main issue of this step was the removal of natural genome: synthetic DNA was trapped out from agarose plugs and digested. The synthetic genome was then transplanted into host yeast. In the discussion authors outlined how was important the quality control of each step. Indeed, none mutations had to be introduced into the synthetic genome, especially in gene crucial for cell viability. Indeed, they explained that lost several weeks because of a mutation in DNA gene. The novelty of this paper is the capability to produce a synthetic genome compatible with cell life and propagation. Indeed, the technology to produce short or longer sequences, i.e. plasmid, is quite common and already commercially available. In this case, yeast still is able to duplicate and the genome is pretty more complex than a plasmid. Informatics skills to predict the correctness of the final genome are essential to successfully accomplish this project. Finally, some ethical concerns about the opportunity to manipulate genes at this point and generate life arise from similar studies. Authors self invite the public opinion and other scientists to continue the debate about ethics. Indeed, we must keep in mind that the final goal of science advances is the human life improvement, in terms of quality, health, environment and similar researches may have important advantages for overall world and us. -
Ten years after the Human Genome Project
Posted on April 6th, 2010 No commentsThe Human Genome Project started ten years ago, with the challenging promise to sequence the whole genome and definitively understand all genetic secrets.
Two astonishing –but also scientifically interesting- surprises were presented to scientists of all countries: firstly only few genes are present in the genome, and this number -20000- is not so different from those of other species; secondarily, the major portion of DNA has regulatory functions rather than encoding significance. Thus, human genome sequencing has generated a lot of further questions about the mechanism of expression tuning. In 1960s Jacob and Monod demonstrated the presence of gene regulator in prokaryotic organisms, such as E. Coli; only few years ago we obtained the confirmation of this presence also in the human genome and numerous gaps have to be filled to reach a comprehensive understanding of molecular mechanism in cells. Epigenetic studies, microRNAs identification and gene expression analysis will help to gain a complete overview of human genome regulation, in addition to gene sequencing. Moreover, the Proteome Project will continue to clarify how proteins are involved in cellular life. Fortunately, a lot of open questions still be unsolved, and a lot of work has to be done by scientists worldwide. -
The IntOGen interface
Posted on March 23rd, 2010 No commentsSometimes, there is a gap between experimental biology and clinical medicine while a continuous interchange would be auspicial to well direct experiments and keep updated the therapies. An interesting tool has been developed at the Barcelona Medical Research Park (Spain).
IntOGen is a frame work that collects, integrates and manages data derived from genome- wide experiments on large scale projects such as the Cancer Genome Atlas and the International Cancer Genome Consortium. Scientists manually annotate all samples by using the International Classification of Disease for Oncology vocabulary, in terms of tumour topography and morphology. Furthermore, they apply statistical methods to identify the most relevant alterations, by analyzing multiple studies on the same kind of tumour. Finally, they consider the role of whole biological modules, such as a pathway, to demonstrate the involvement of a single gene altered. The website www.intogen.org is available for free and allows to know modules and genes important in cancer, share experiments and analyze data in the context of cancer. This interface has been built to fill the gap between medicine and molecular biology. Similar tools should be really useful not only for cancer but also for other kind of diseases, such as neurodegenerative disorders. -
Novel role of ABA in plant signaling
Posted on December 18th, 2009 No commentsAbscisic acid is a small molecule essential for plant life because it regulates seed maturation and bud dormancy as well as stressors response, such as high salinity of environment or extreme temperature. Giving the peculiarity of this substance in plant life, several studies have been performed to clarify the mechanism of action and better understand how it could be possible to use it to introduce useful qualities into plant genome.
ABA works as transcription regulator by acting on soluble receptor. In absence of this plant hormone, the receptor is usually bound to a phosphatase that blocks its activity by inhibiting the associate kinase; ABA determines a conformational change allowing the relieve of kinase inhibition; this kinase can phopshorylate the receptor and activate the transcription. Crystal structures of ABA receptor are available in different states, thus describing the details of this mechanism. As it has been proposed also for other plant hormones, also ABA seems to enhance critical protein- protein interactions and modulate post translational modifications such as ubiquitylation or phosphorylation as in this case that regulate plant cells. Plant hormones execute several important functions in plants; the control of these biochemical pathways could generate important advantages for agriculture and plant productivity. -
New perspectives on botanic sciences
Posted on December 16th, 2009 No commentsPlant biology has acquired a great interest in biotech company, because of the opportunity to modify plant genome and introduce genetic improvement. Of course, selecting the best types and matching them to obtain more productive species is a current practice since long time. We remember how Mendel performed his studies about genetics and hereditary law, matching different plants of pees in order to observe how phenotypical characteristics were transmitted to progeny.
So, how to match plants is an ancient knowledge for us. Now the diffusion of molecular biology techniques allows to fast the matching process and directly introduce genetic alterations in the exact point of genome. This new approach has the great advantage to be specific, fast and all the progeny has the new gene and the new quality. Modern botanic sciences can also use biochemical knowledge to activate or block signalling pathways and have new properties of plants in reversible manner. This could be useful to increase the productivity only in a limited period of the year, for instance when the environmental conditions are more favourable. In conclusion, molecular biology and biochemistry have a revolutionary impact also in ancient sciences, such as botanic, that have accompanied humans since the beginning. -
Databases
Posted on November 20th, 2009 No commentsTwo main databases are now available: the EMBL-EBI and the NCBI for Europe and US, respectively. These two databases are connected and all the information present are available in both systems. Another database is provided by a Japanese laboratory and is online at genome.jp.
Main databases contain information about DNA sequence, two examples are EMBL datalibrary and GenBank; all other databases regarding RNA, proteins and polymorphisms or rare diseases are connected to these two ones. Databases are usually checked by operators or software: the difference between these two control systems could be observed in the redundancy because manual control is usually more systematic than these performed by software. About proteins, three secondary databases are currently used: Swiss-Prot, TrEMBL and PIR. In these websites several bioinformatic tools are available to align sequences, predict primary and secondary structure of proteins or determine the isoelectric point, all these information are important especially at the beginning of the study. Other databases like PDB or Modbase offer three-dimensional structures of proteins and prediction of three-dimensional structures, respectively. As well as PROSITE collects information about protein motifs, functional domains and so on. Last but not least, Genome.jp is preparing a new tool, KEGG pathway, useful to retrieve information about enzymes and metabolic pathway. Good work! -
Manipulation of bacterial genome in yeast
Posted on October 28th, 2009 No commentsEven if manipulation of bacterial genome is often difficult and challenging, engineering allows to better understand bacterial biology and genetics. Researchers from C. Venter Institute improve a protocol to clone bacterial genome in yeast, manipulate it and boot it up in bacteria self. To do this they chose an “easy” model, Mycoplasma, because this organism doesn’t have bacterial wall, its genome is small and A-T rich, so is more properly replicated in yeast than ones rich in G-C. Furthermore Mycoplasma has non-standard genetic code that can not be translated in yeast, preventing the synthesis of bacterial proteins toxic for yeast.
What did scientists perform to achieve this important result? They cloned Mycoplasma genome into yeast artificial chromosomes (YACs), genetically manipulated it and then transplanted it into the final organism receiver. Two concerns could prevent this goal: one was the possibility that restriction endonucleases recognised foreign sequences and degraded them and the second one was that yeast modified bacterial genome. Fortunately this last event didn’t occur, while to limit endonucleasic activity, scientists hypermethylated donor genome and eliminated endonucleases from receiver organism. This protocol could be improved in order to become a conventional technique for bacterial manipulation in order to have another tool to solve human needs in medicine and environmental preservation. -
High-throughput re-sequencing to identify rare allelic variants
Posted on August 12th, 2009 No commentsRecent advances on genomic studies allow to re-sequence genes with high accuracy and in high throughput procedure. Rare allelic variants are important to be analyzed because are often the molecular basis of human disease. Indeed, numerous syndromes are associated to mutations in rare allele, but a lot of work have to be done yet.
A new protocol has been published to apply an array to high throughput re-sequencing. Two problems were encountered at the beginning of the study: the upstream target preparation techniques available until now were not able to produce thousands of samples simultaneously and the accuracy was too low to distinguish rare variant to false positives. Scientists from Genentech and Stanford Genome Technology Center developed a method for target amplification by capture and ligation (TACL) based of novel probes for genomic DNA that are amplified by PCR, incorporate deoxyuridine and are purified. TACL method provided high reproducibility ad specificity in terms of capturing of the target regions also when the starting sample concentration was lower than 15 nanograms. Then, they cloned TACL probes into bacteria and used them to hybridize to probes previously captured. The bacterial growth in selective media allowed to recognize where the mismatched was preset, thus enriching the rare alleles. This technique seems promising and user-friendly because doesn’t need of particular instrumentation to be successfully employed.
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Overview about the transposon – based technology
Posted on August 10th, 2009 No commentsTransposition is a new approach to genome manipulation. Transposable elements are DNA segments with the peculiar capability to move about the genome. Transposon-based genetic strategies are applied for the transgenesis of somatic or germ-line cells, for insertional mutagenesis, both loss or gain of function and for non viral DNA transfer into cells in cell-based clinical applications. Transposons have been identified in all organism and have been distinguished in two classes in relation of their mechanism of action. The mobility of class I elements, also named as retro-transposons, work through RNA intermediate and encode for one nucleic binding protein and an enzyme that acts as endonuclease and retro transcriptase: endonuclease generates a nick into the target DNA and retro transcriptase starts the reverse transcription of the RNA of the transposon from nicked DNA.
Class II transposable elements are simpler than those of class I and their genome, flanked by two inverted terminal repeats, encodes for a transposase protein that allows the excision of transposon and the insertion into DNA target. In this case, the transposition process could be easily controlled by separating the transposase from the transposable DNA. Several kind of transposons are now available and are used as a vector to manipulate the genomes: the first studies were performed on C. Elegans and Drosophila and only since 1997 when has been demonstrated the re-activation of the Sleeping Beauty transposon system, has been employed also in vertebrates and mammals. Parameters that have to be considered during the choice of transposon as a vector are the size of DNA that could be moved and the integration site of preference. The capacity of moving large DNA fragments varies between the species of transposons, Sleeping Beauty transposon is inhibited by large size fragments, by contrast piggyBac transposons are more tolerant to large size without reducing their efficiency. The insertion point of transposons is non-random, but occurs in hot regions of the genome, for instance intron or transcription unit, that are characteristic of each type of transposon. The integration site preference is important for the application of transposon vectors: for example, mutagenesis screening could be more efficient by using elements that tend to land in genes, while human gene therapy protocols could require vectors showing the least preference to target gene. Transposons are used to perform recessive genetic screens in embryonic stem cells and in germ-line in vivo and to transfer DNA into stem cells and oocytes and embryos. In the next future, the transposon- based technology could be applied on germ-line transgenesis of laboratory animals and in larger species, like sheep and pig. Furthermore, gene transfer could be improved into therapeutically relevant primary cells, including stem cells, allowing the implementation of ex vivo and in vivo therapies. In conclusion, transposon approaches seem to be really promising for future advances of science.
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