Since the advent of dideoxy sequencing method by Sanger, important biological data has accumulated explosively. Several automations to this method, has made sequencing routine work in molecular biology research. But several new sequencing technologies do emerge in aim of reducing cost and time further. One such technology is Nanopore sequencing technology.
Nanopore is simply a hole and the internal diameter is in the order of 1 nanometer ranges. When two chambers containing different electrolyte concentrations are separated by a nanopore containing membrane a potential difference is formed between the two chambers. If this voltage is disrupted (say for example by blocking the pore), the resulting ionic current can be measured using standard techniques. Thus it was independently proposed by two researchers from University of California and University of Harvard, that the modulation of this current could also be done by electrophoretically driving a strand of DNA or RNA through a nanopore of appropriate diameter. If the internal diameter of the nanopore is approximately equal to a nucleotide of a ssDNA molecule, two important properties could be achieved.
1. The unraveling of coiled DNA upon mobilizing through the nanopore
2. Translocation of a single nucleotide in the nucleic acid molecule through the pore in sequential order
Due to the partial blockage of current through the nanopore upon nucleotide translocation, there is a relative reduction of ionic current compared to the current through the nanopore when it is empty. Thus each nucleotide in the nucleic acid molecule produced a unique current modulation upon passage through the nanopore. However several problems persist given the extraordinary ability of nanopores for sensing single molecules.
Solving all drawbacks, Oxford Nanopore technologies has introduced the world’s first miniaturized 900$ sequencing machine. It may be possible to sequence a whole human genome in just hours, which took 13 years less than a decade ago.
1. http://www.nature.com/naturebiotechnology – The potential and challenges of nanopore sequencing
African sleeping sickness or African Trypanosomiasis is a disease caused by the trypanosoma parasite which is biologically transmitted by the tsetse fly (Glossina sp.). The insect is essential for the completion of the parasite’s life cycle. Individuals with the disease exhibit symptoms in two stages. First stage being the haemolymphatic phase and the second stage being the neurological phase. The disease is known as the sleeping sickness due to the second phase which includes symptoms such as confusion, lack of coordination and disrupted sleep cycle.
Sodalis glossinidius is a gram-negative endosymbiotic bacteria living in the alimentary canal of the tsetse fly. These bacteria help the tsetse fly to acquire nutrients that are not synthesized by the fly or not present in their diet. The striking feature of S. glossinidius is that it is one of the few endosymbiotic bacteria that could be cultured and modified genetically.
A group of scientists from Belgium have utilized this feature to genetically modify the endosymbiote to express proteins that can control the sleeping sickness parasite. This is known as paratransgenesis. A transgene is introduced into S. glossinidius where the gene products interfere with the development of the pathogen inside the tsetse host and at the same time preserving host and the symbiote’s viability and fitness.
Nanobodies® (Nbs) are the smallest known antigen binding fragments that target specific antigenic epitopes that are less efficiently targeted by antibodies. Nbs have several superior properties such as small size and stability and therefore are perfect candidates to be used in paratransgenesis. A trypanosome surface glycoprotein specific Nb_An33 Nanobody® was used for targeting the trypanosoma parasite.
Expression plasmids were constructed using the Nbs gene (Nb_An33) with a lac promoter and two genes for the secretion signal for extracellular release of Nb_An33 Nanobody®. The Nb_An33 secreting strains exhibited growth comparable to the wild type. Expressed Nanobody concentrations were determined using ELISA. Binding ability of the extracellularly expressed Nb_An33 with trypanasoma parasite was determined using flow cytometry and fluorescence microscopy.
These results indicate that S. glossinidius could be manipulated to be a paratransgenic organism and the use of Nbs effectors in biological control of other vector borne diseases.
Source : Microbial cell factories
It is a known fact that half of our chromosomes are obtained from our mother and the other half from our father. These chromosomes contain genes. Some of these genes are expressed only from one of the two inherited parental chromosomes which is known as genomic imprinting. Some imprinted genes are maternally inherited and some are paternal. This is one of the modes of gene regulation that mammalian genes utilize to control gene expression. Evidence exists for genomic imprinting to occur in plants as well.
One evidence for imprinting comes from experiments conducted in mice where mice embryos containing two maternal sets of chromosomes (both set of chromosomes from the mother and no contribution by the father) or vice versa were in viable.
Some imprinting disorders are identified in humans too. Loss of paternally inherited chromosome 15 results in Prader-Willi Syndrome (PWS) and loss of maternally inherited chromosome 15 delivers Angelman Syndromes (AS). Both the diseases are distinct neurological disorders.
All imprinted genes identified to date are expressed in the developing embryo, placenta and in the brain of the fetus and the newborn.
Imprinted genes in two parental chromosomes are tagged or marked for transcriptional machinery to distinguish them from non imprinted counterparts. These marks are known as epigenetic modifications that include DNA methylation and post translational modification of core histones. These modification alter chromatin structure and influence transcription.
One rather startling information suggests a higher percentage of imprinted disorders in people born via in vitro reproductive technologies. Research continues to reveal further insights into this exquisite mechanism of gene regulation.