samedi 3 avril 2010
mercredi 23 avril 2008
Phage display
Phage display is a test used to screen for protein-protein interactions and protein-DNA interactions by integrating many genetic sequences from a DNA library into the genome of a suitable phage.
Phage display animation tutorial
vendredi 7 mars 2008
DNA Microarray Methodology - Flash Animation
The following link shows a very interesting animation about the DNA microarray methodology:
DNA Microarray Methodology - Flash Animation
DNA Microarray Methodology - Flash Animation
samedi 1 mars 2008
Epigenetics
Epigenetics refers to the study of different mechanisms controlling gene expression independently from the DNA sequence. In fact there are heritable modifications in gene expression, going from one generation to another one, which are not caused by what is coded in the genome itself.
The role of epigenetic factors might be an extra control on gene regulation, in cell differentiations and cell developments. Potentially epigenetic abnormalities could be causative factors in cancer, genetic disorders, autoimmune diseases and aging.
DNA methylation and histone modifications are 2 basic principles of epigenetics. Actually when methyl groups attach to the histones, they can affect the expression of the DNA's genetic code, by turning special genes on or off. Distinct methylation and histone patterns exist in every cell, constituting a sort of second genome, the epigenome.
The role of epigenetic factors might be an extra control on gene regulation, in cell differentiations and cell developments. Potentially epigenetic abnormalities could be causative factors in cancer, genetic disorders, autoimmune diseases and aging.
DNA methylation and histone modifications are 2 basic principles of epigenetics. Actually when methyl groups attach to the histones, they can affect the expression of the DNA's genetic code, by turning special genes on or off. Distinct methylation and histone patterns exist in every cell, constituting a sort of second genome, the epigenome.
Image from the following article:
Phenotypic plasticity and the epigenetics of human disease
Andrew P. Feinberg
Nature 447, 433-440(24 May 2007) doi:10.1038/nature05919
vendredi 29 février 2008
RNA Interference
One of the most revolutionary discoveries in molecular biology in the past decade may well be the discovery of RNA interference (RNAi); a mechanism in the cell biology of many eukaryotes in which fragments of double-stranded ribonucleic acid (dsRNA) interferes with the expression of a particular gene whose sequence is complementary to the dsRNA. RNAi has been applied as an experimental technique to study the function of genes both in vivo and in vitro. Researchers are now trying to use this technology for therapeutic purposes in human disease.
RNAi is an RNA-dependent gene silencing process that is mediated by the RNA-induced silencing complex (RISC). The process is initiated by the RNase-III-like enzyme Dicer, which binds and cleaves long dsRNA to produce short double-stranded fragments with two nucleotide 3’ overhangs on either end, called small interfering RNAs (siRNAs). siRNAs are coupled to RISC complex then binds to a target mRNA in a sequence specific manner by base-paring and induce mRNA target cleavage. The cleaved RNA can be recognized by the cell as being aberrant and then destroyed. Therefore without mRNA there is not any translation to get proteins afterwards. This technique is also referred sometimes as gene “knockdown” as RNAi may not totally destroys the expression of the gene but just somehow turns the genes off , unlike the classic processes of gene “knockout” in which expression of a gene is completely eliminated by removing its DNA sequence.
The great power of RNAi in silencing the gene expressions with high specificity has made this technology an interesting subject for many recent studies, trying to use RNAi as a treatment for HIV, viral hepatitis, cancer and genetic diseases. However the therapeutic applications of this method has not yet been approved and still there is considerable difficulties, including difficulties in efficient delivery and uncertainty about potential toxicity of this method , which are left to be overcome in order to apply in vivo. Also potential side effect of using cellular machinery for directing sequence silencing is the big question that is not answered yet.
The following animation shows the RNAi process. It is from the Nature Reviews RNAi collection:
mercredi 27 février 2008
Western blot
Western blot (also called immunoblot) is a technique to detect specifically one protein in a mixture of large number of proteins and to obtain information about the size and relative amounts of the protein present in different samples.
In this method first proteins are separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Then they are moved onto a nitrocellulose membrane. The proteins retain the same pattern of separation they had on the gel.
The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. This step is required as both antibodies and the target are proteins and proteins like to bind to nitrocellulose. By placing the nitrocellulose membrane in a dilute solution of protein, we can prevent the unwanted interactions between the membrane and the antibody used for detection of the target protein as the protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached.
An antibody is then added to the solution which is able to bind to its specific protein and forms an antibody-protein complex with the protein of interest. (In fact there is no room on the membrane for the antibody to attach other than on the binding sites of the specific target protein).
Finally the nitrocellulose membrane is incubated with a secondary antibody, which is an antibody-enzyme conjugate that is directed against the primary antibody.
The location of the antibody is revealed by incubating it with a substrate that the attached enzyme converts to a product that can be seen and followed and then photographed.
Western blot is a confirmatory test for a positive HIV ELISA as it allows one to visualize antibodies directed against different viral protein in a human serum sample.
In this method first proteins are separated using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Then they are moved onto a nitrocellulose membrane. The proteins retain the same pattern of separation they had on the gel.
The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. This step is required as both antibodies and the target are proteins and proteins like to bind to nitrocellulose. By placing the nitrocellulose membrane in a dilute solution of protein, we can prevent the unwanted interactions between the membrane and the antibody used for detection of the target protein as the protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached.
An antibody is then added to the solution which is able to bind to its specific protein and forms an antibody-protein complex with the protein of interest. (In fact there is no room on the membrane for the antibody to attach other than on the binding sites of the specific target protein).
Finally the nitrocellulose membrane is incubated with a secondary antibody, which is an antibody-enzyme conjugate that is directed against the primary antibody.
The location of the antibody is revealed by incubating it with a substrate that the attached enzyme converts to a product that can be seen and followed and then photographed.
Western blot is a confirmatory test for a positive HIV ELISA as it allows one to visualize antibodies directed against different viral protein in a human serum sample.
mardi 26 février 2008
La Rétrotranscription de l'ARN des Rétrovirus
La Rétrotranscription (the reverse transcription) est une étape importante dans le cycle infectieux des rétrovirus. Elle consiste en synthèse d'un ADN proviral à partir du complexe d'initiation formé d'une amorce ARNt hybridée à l'ARN génomiques des rétrovirus. La rétrotranscriptase, l’enzyme impliqué dans ce processus, est une des principales cibles des médicaments antiviraux.
Dans le film j'ai essayé de montrer les différentes étapes de la rétrotranscription:
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