Bioinformatics and Genomics News and Views » DNA

The whole genome sequencing to identify Mendelian disorders

Jessica P. — Wed, 09 Jun 2010 10:07:53 +0000

The 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.

Cellular transfection

Jessica P. — Mon, 17 May 2010 12:21:09 +0000

Experimental research uses modern and old cellular biology techniques for an high number of applications. For this reason, cellular biology shows great advances in last decades. Improvement in cell culture media allows propagating cell lines that was not possible to culture just few years ago. For instance, stem cells are now available and can be maintained in plate without loosing their stemness. Despite these advances in cell culturing, the method of transfection, most widely used, still is the calcium phosphate method. Indeed, either adherent and in suspension cells can be treated with this technique and can be successfully transfected. The general protocol requires the direct addition of calcium phosphate DNA complex to cell medium, at least for six hours. Nevertheless, some modifications have been proposed in order to increase transfection. It could be possible to pre-treat cells with chloroquine, that is able to produce pores into cellular membranes. Otherwise, it’s also possible to shock cells with glycerol for less than two minutes. After glycerol treatment, it’s fundamental to accurately rinse cells with sterile PBS in order to completely remove the glycerol self. The calcium phosphate method is also inexpensive because all reagents can be produced in house, by using salts and powders usually present in research lab.

Protein macroarray

Jessica P. — Tue, 20 Apr 2010 11:00:44 +0000

Protein macroarrays are chips of gold or silver where proteins are bound and
analyzed. Several methods have been developed to attach the proteins on chip surface without interfering with their folding or functionality. In some cases, it has been useful to use particular tags at the N or C terminal of proteins of interest to facilitate the binding. The SNAP tag is one of common tag used for this purpose. This tag is based on the fusion of your protein to a small mammalian protein, the O6-alkyl-guanine- DNA- alkyl-tranferase (AGT). AGT can covalently bind a benzyl group from its substrate, resulting in a self labeling process with extremely low background. In macroarray chips, the SNAP tag can contribute to stabilize the binding through a covalent interaction. Macroarrays will be really important tools to understand the mechanism of protein- protein interaction, ligand receptor interaction as well as drug to target interaction. After the proteome project, it’s crucial that also technology will be updated to obtain quantitative results on protein functionality. The main fault of protein macroarray is the high expense to produce chips; thus it will be necessary to find out new solutions to reduce costs, and the use SNAP tag may be one of those.

Replication timing analysis

Jessica P. — Thu, 08 Apr 2010 11:00:27 +0000

The replication timing assay is a current lab method used to determine the exact phases of replication process. Exponentially growing cells are labelled with 5- bromo 2-deoxyuridine to mark newly generated cells. Indeed, the bromo-deoxyuridine is incorporated into DNA in place of thymidine. Fluorescent dye DAPI stains the whole cellular DNA and enables to fractionate cells on the basis of total amount of DNA and thus, the cells’ stage of the synthesis phase of cell division.
Then, labelled DNA is isolated by immunoprecipitation and analysed by PCR on the loci of interest. A recent paper published this year in PNAS, proposes to substitute the PCR with the DNA assembling into separate Illumina sequencing libraries and sequence it. It seems that the most of the genome is going to be replicated at about the same time in different cell types, but the study of other cells populations will be clarified the timing. Furthermore, sequencing will permit analysis also of rare cell populations because of the low number of cells required for the assay. In conclusion, this new approach of replication timing assay allows extending many analyses at the whole genome levels, improving the throughput and accelerating scientific advancement.

Ten years after the Human Genome Project

Jessica P. — Tue, 06 Apr 2010 11:52:32 +0000

The 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.

Application of molecular biology to medicine

Jessica P. — Fri, 19 Mar 2010 14:08:48 +0000

Modern medicine is based on evidence. Despite few years ago, physicians based their diagnosis and therapies on their previous experiences, now clinical trials, approved protocols and worldwide accepted treatment help physicians to make the better choice for their patients. To reach this important goal, molecular medicine gave a big contribution. Molecular medicine is a novel branch of medicine in which molecular biology or biochemistry techniques are usually used to accomplish exams and screening or diagnose the diseases. Molecular biology labs have worked as research and development department to improve protocols or set up novel methodologies at the beginning just to be used for research purposes, then applied to diagnosis. For instance, we could focus our attention on the polymerase chain reaction, namely the PCR.
This techniques has been developed to produce DNA portion in vitro and has quickly revolutionized molecular biology, allowing cloning, sequencing and in general gene manipulation. The improved type of PCR, the real time PCR is now currently used in modern hospital to detect certain diseases, such as recidivate leukemia in patients’ blood or gene signature in familial diseases. Other important tools which have found a good application in diagnosis are the monoclonal/ polyclonal antibodies. Several techniques employed antibodies to detect proteins in cellular lysates through western blotting, or directly in whole cells, through the immuno-chemistry and molecular imaging. As well as, it’s also possible to purify small amount of native protein through immuno-precipitation and so on. Antibodies are really largely used in biochemistry labs and some techniques are applied also in diagnostic labs.
For instance, we can talk about the ELISA assay, normally used to quantify serum proteins or cell sorting analysis, in which some antibodies that specifically recognize surface proteins are used to separate different kind of cells. Finally, also cellular biology gave important results in modern medicine. The capability to culture in vitro cellular populations changes the therapeutic opportunity for a lot of diseases. For instance, now it’s possible to select healthy cellular population in leukemic patient, propagate it in vitro and then draft it, reducing in this way several problems of rejection and immuno-suppressive events. We can continue to talk about other examples of molecular, biochemical or cellular protocols that have found a great application in clinics. This overview strongly confirms how scientific advances are important because they have an immediate benefit on modern medicine and as a consequence on the human life. It’s important to remember that not only good protocols, but also good quality of science and good data management and reporting are two further parameters to improve modern medicine, but this is the topic for another post!!!

Role of natural antioxidant elements in cardiovascular disease prevention

Jessica P. — Sat, 27 Feb 2010 15:55:01 +0000

Natural antioxidant elements consist of carotenoids, vitamins, mineral salt contained in food. These components are important to remove free oxygen derived compounds that are highly reactive and generate lipid peroxidation and DNA damage. Lipid peroxidation is one cause of lipoprotein modifications. In particular low density lipoproteins (LDLs) are intensively modified by free radicals and several forms of altered LDL have been identified.
The overall effect is an increasing likelihood to produce atherosclerotic lesions, followed by an increased risk to have cardiovascular problems. In vitro some evidences about the ability of natural antioxidants to remove free radicals have been observed, but it’s not totally clear what is the significance in human, because clinical studies still are controversial. Even if some studies demonstrated a protective role of natural antioxidants against cardiovascular diseases, it’s quite difficult to evaluate the exact concentration of these components in the serum, and few dose- response studies are available up to date. Vitamin E seems particularly involved in cardiovascular disease protection because low levels of this compound have been measured in patients who had stroke or other cardiovascular problems. Unfortunately, prospective study is a limited tool to evaluate the efficacy of vitamin supplement as a tool to prevent heart failure. Further analyses must be done to ascertain the role of antioxidants in disease prevention; at this time we can still eat a lot of fruit and vegetables for our pleasure.

Mutational analysis of promoter region

Jessica P. — Wed, 17 Feb 2010 15:08:04 +0000

Promoter region is the regulatory portion of genes that controls transcription. Even if this portion is crucial for finely tuning protein expression, and is known that epigenetic alterations are dangerous for this control, poor knowledge are available about the role of point mutations. Indeed, several point mutations are identified in promoter region, their biological consequence is poorly understood and studied. Scientists of the University of Washington set up an array to screen the effect of mutation on promoter functionality. They analysed all possible point mutations into a core promoter: the construct presented the native sequence, followed by mutated promoter and the transcription fragment. Each construct was codified by a barcode; thus, after transcription, a quantitative measure of transcriptional activity was obtained. In this way, all mutations were screened and quantitatively characterized. A surprising result was that one mutation generated down-regulation of the expression, while two mutations didn’t alter so much the transcriptional activity. A possible explanation was that the interaction between DNA and polymerase was stabilized in presence of one mutation, reducing the capability to move onto DNA molecule. This experiment was performed in vitro with cellular extract. Scientists knew that the best way to accomplish this screening is to use transfected cell lines, and this will be the possible next step.

New perspectives on botanic sciences

Jessica P. — Wed, 16 Dec 2009 10:45:50 +0000

Plant 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.

Direct to consumer genetic services

Jessica P. — Fri, 04 Dec 2009 12:00:59 +0000

Large expansion of direct to consumer genetic services is observed world-wide. This service offer single gene tests as well as screening of customer’s DNA at various loci to identify some polymorphism associated with certain disease. For instance, in women with familiar past history of breast cancer can be monitored for BRCA1 and BRCA2 genes, that are recognized as elevated risk factors. Furthermore, some genes involved in Alzheimer’s disease can be tested. A study was performed to evaluate the consequences of knowing the genetic makeup and the possible disease encountered in the future: it seems that knowledge about genetics doesn’t influence life, for instance by generating depression or anxiety. More concerns are about the scientific value of these tests, the modality to perform them and the clinical utility. Different approaches have been done by government to regulate this modern phenomenon: for instance, in Germany these tests are forbidden and only physicians can suggest genetic test carried out in appropriate institutions. In United Kingdom it has been proposed a self- policing by industry. In the US regulation is up to Federal states and varies largely. First implication of the large diffusion of this kind of test is the increase of analysis and examination to confirm genetic information. Medical intervention can not be the direct consequence of the test, but more deepened talks with physicians and genetic counsellors should be preferred.