Epigenomic Profiling Reveals DNA-Methylation Changes Associated with Major Psychosis

Epigenetics can be defined as changes in gene transcription through modulation of chromatin, which is NOT brought about by changes in the DNA sequence.Schrizophrenia(SZ) and bipolar disorder(BD) both conditions are known to be etiologically related and both conditions together are termed major psychosis. Different studies previously on major psychosis are based on interplay between genetics and epigenetics and observations led to the identification about the importance of epigenetic factors. Until now few studies on the role of DNA methylation in major psychosis are done but none have taken a whole genome wide epigenomic approach.In this paper author investigates the whole genomic approach using the DNA from frontal cortex, a region which has been etiologically implicated in psychiatric conditions. Samples from both SZ and BD patients were analysed with comparison to control samples.
Authors examined the DNA methylation with two complimentary approaches. They performed a microarray epigenomic scan using CpG island microarrays after the enrichment of unmethylated fraction of brain DNA by using restriction enzyme HpaII and McrBC.Secondly they performed a hypothesis driven analysis of DNA methylation for priori evidence of the role in the etiology of the major psychosis. They also did a CpG island microarray to investigate the differences in the germline DNA in BD patients. Numerous loci including glutamatergic and GABAergic neurotransmission, brain development and other processes linked to disease are analysed. Methylome network analysis revealed the decreased epigenetic modularity in the affected individuals.Hence considerable epigenetic changes were found as evidence for the epigenetic relation to etiology of major psychosis. As the epigenetic changes are reversible this can lead to epigenetic treatment of the psychiatric conditions. Epigenetics is also found to have role in memory, hippocamal development and many other functions of the brain and hence the study of epigenetics in the psychiatry field might uncover many unknown facts of brain chemistry.

Raja Lella
41827581

A method to regulate protein function in living cells using synthetic small molecules

Nowadays, a lot of techniques for studies of gene expression and function are at DNA or mRNA level relying on a major strategy of perturbing a target gene expression and observing the change of phenotype. However, techniques that can regulate target proteins directly at protein level are required because of their higher efficiency. A rapid and reversible technique for protein function regulation in living cells is developed recently. It can change the stability of a target protein by expressing fused to mutated human FKBP12, a destabilizing domain, which can lead to degradation of the entire protein. The stability of the fusion protein can be control by attendance of a synthetic small molecule, Shld1, which can bind to FKBP12 and stop the degradation. This regulation is dose-depended and reversible. Therefore, predictable change of phenotype can be achieved by control the concentration of Shld1. In addition, this technique can be widely used in common cells lines to regulate various proteins.

Hu Lingbo
41943904

PCR-free method detects high frequency of genomic instability in prostate cancer

Since the introduction of Polymerase chain reaction (PCR) techniques, it has been the method of choice for many tumour instability studies. PCR-based methods are useful for the study of small-scale somatic alterations. However, these methods often underestimate the frequency of mutations in heterogeneous tumour genomes. Thus in such cases, sequencing of individual clones may prove more fruitful. The paper by Makridakis et al (2009) investigates the use of a random cloning/sequencing method to identify genomic instability in prostate cancer. This method involves the random cloning of DNA into plasmid vectors, and the subsequent transformation of competent E. coli cells, for a non-PCR based method of cloning. Whilst cloning DNA using plasmid vectors is not a new technology, the combination of random cloning and sequencing of DNA for the use of genomic instability studies is a novel idea. Using this random cloning/sequencing method the authors reported findings that were several orders of magnitude higher than those reported previously.

Emily Chan

MAGE

Multiplex automated genome engineering (MAGE) is a break through technology that has been introduced recently in August 2009. It is a major improvement to the current technology of genome engineering. MAGE is capable of modifying genomes on a large scale and product about 15 billion different genomic patterns in just 3 days. This scale of genome modification would usually take years to achieve. Unlike current technologies available, MAGE is highly efficient, inexpensive and automated, which means more companies can afford it and lesser man hours is required to generate the equivalent number of mutations.

MAGE have been tested for the production of Lycopene in the E.coli strain EcHW2. By targeting 2 sets of genes within the genome of the bacteria, they were able to carry out gene mutations on a large and random scale before isolating 6 colonies that contained variants with amplified production of lycopene as compared to the wild type strain E.coli. These colonies were chosen because they produced intense red pigmentation.

MAGE can generate different levels of diversity by tuning 3 factors. The degree of sequence variation at each locus, the number of loci target, and the number of MAGE cycles. Therefore the diversity of modification that can be generated is adjustable and is highly specific. This technology could eventually be used to produce industrial chemicals, drugs, fuel and anything else that comes out of bacteria

Wilson Leow
41552762

Label-less probes for microarrays - Nano-scaled Polystyrene films

Blog Post for Second BIOC6006 Presentation
Microarray Analysis using disiloxyl 70mer oligonucleotides
Microarrays are one of the most powerful tools in modern biology. A single chip, containing thousands of probes can provide data on the regulation of thousands of genes. A microarray works via the principle of hybridization.
Each microarray chip contains hundreds to thousands of tiny holes. In each hole (or spot) is a cDNA (or sometimes cRNA) sequence from a particular gene. The cDNA is can be taken from a wild type organism living in normal conditions, as a background in a comparison-type experiment, or it can be synthesized. This background or synthesized cDNA (or cRNA) is labelled (usually with a fluorescent, red probe) and placed into the wells. cDNA or cRNA is obtained (by Reverse Transcription) from the organism when it has been exposed to the conditions we are testing. These probes are labelled with a different kind of probe (usually a fluorescent green probe). The cDNA (cRNA) taken from the test organism is mixed with the spots containing the background cDNA or synthetic probes. They are placed in conditions in which the test organisms’ cDNA will hybridize with the probes already in the well. All non-specific stuff will wash off.
After this is done, each well will have an amount of background or neutral cDNA (cRNA) and an amount of cDNA (cRNA) from the organism that was tested. If the gene was more upregulated in the neutral/background organism, then the spot will glow red. If the gene was more upregulated in the test organisms, then the spot will glow green. If the gene is regulated in a similar manner in both, then it will glow yellow.
In this paper “Label-less fluorescence based method to detect hybridization with applications to DNA micro array”, by Sanjun Niu, Gaurav Singh and Ravi. F. Saraf, examines (as the title will tell you), examines how it is possible to make DNA probes fluorescence without having to add fluorescent labels, which takes time, and money and could interfere with the functioning of the probe.
By analysing how immobilized ssDNA probes scatter light, the group were able to create a fluorescent polystyrene film , whose thickness is determined at the nano-meter scale, that the probes will be immobilized to. Binding of DNA to the probes will create dsDNA and change the scattering of light, creating a fluorescence contrast which is extremely sensitive. The team calculates that it will increase the detection of binding by an entire order.

By Mark Phillipps
40995867

Polony Multiplex Analysis of Gene Expression (PMAGE)

Several methods have been developed to define the complete repertoire of RNA transcripts including microarrays and serial analysis of gene expression. However many of these fail in detecting rare transcripts or mRNA at low expression and hold with them multiple limitations. Polony multiplex analysis of gene expression (PMAGE) is developed to detect mRNA as rare as one transcript per three cells. One sequence tag from each mRNA molecule is amplified onto a separate 1-micrometer bead, denoted as polymerase colony or polony. Approximately 5 million polonies are arrayed in a flow cell for parallel sequencing. One of the applications of this method is identifying early transcriptional changes that preceded pathological manifestation of hypertrophic cardiomyopathy in mice carrying a disease-causing mutation.

Posted by Uyen Tran
ID: 41987494

Solexa Sequencing Technology

The Illumina Genome Analysis System is a groundbreaking new platform for sequence analysis and functional genomics. Dramatically improving speed and reducing costs, it is suitable for a wide range of applications including whole genome and candidate region sequencing, expression profiling, DNA-protein interactions, and small RNA identification and quantitation. Leveraging proprietary reversible terminators and Clonal Single Molecule Array technology, the Illumina Genome Analysis System can generate several billion bases of data per run and in the process transform the way many experiments are devised and carried out.

By
Crystal D'souza.

MAGE to be the Gauge as we usher in new Page in Molecular Genetics Research?

The lure of the Human Genome Project had given the impetus for the advancement of high-throughput DNA sequencing technology. But what if your game is modifying genomes? Then you are in for a long day, with comparatively ancient technologies where you have to introduce single DNA constructs. This was the case until a group of researchers invented a method called the multiplex automated genome engineering (MAGE).
MAGE not only has a snappy acronym but is able to perform large-scale programming and evolution of cells. MAGE involves a process that targets many locations on the chromosome to modify a single or a population of cells simultaneously. Because this process is cyclical and scalable, it can be automated to produce mismatch, insertion or deletion genetic changes rapidly and continuously.
How is this done? SsDNA or oligonucleotides (oligos) is directed to the lagging strand of the replication fork during DNA replication in E. coli to cause allelic replacement. The oligos are mediated by Bacteriophage λ-Red ssDNA-binding protein β. Sequence diversity is ensured by repeatedly doing this process.
At optimized levels new genetic modifications are introduced in >30% of the cell population in 1 cycle (2-2.5 hours). The efficiency can be controlled by how homologous of the oligo to the chromosome target or in the case of deletion, the size of the deletion. Just to give an idea, at the end of 5 cycles an average change of 3.1 bp per cell across the population can be achieved, which equates to 4.3 X 109 bp variations/day.
What does all this mean? As an example of the power of this method, MAGE was used to modify gene expression to increase the yield of the red pigment, lycopene. Only 20 primary endogenous genes as well as an additional 4 genes through secondary pathways are known to affect lycopene yields. Previous molecular genetics methods have a limitation on the number of genetic components that can be manipulated at a time. With MAGE all 24 genes were manipulated and 15 billion genetic variants were produced in 35 cycles. Through this, genetic variants were identified that gave up to a 390% increase in yield compared to the wild-type yields
Not a bad result to achieve without having to be in the lab for 3 days.

Reference:
Wang, H. H., F. J. Isaacs, et al. (2009). "Programming cells by multiplex genome engineering and accelerated evolution." Nature 460(7257): 894-U133.

Reuben Durairatnam
42140867

A new transgene reporter for in vivo magnetic resonance imaging

The ability to image the gene expression in vivo is important for biological and medical studies, especially in a non-invasive manner. To indicate where and when the therapeutic gene goes is a very crucial step in genetically medical applications. Magnetic resonance imaging (MRI) is a widely used diagnostic tool. It is not invasive, and it provides a good spatial resolution. More importantly, it allows us to view into the opaque subjects.

Therefore, the novel transgene reporter for MRI application in vivo is important because this technology can be used to monitor the gene expression in many kinds of tissues and it is optimal for medical applications such as visualizing preclinical therapeutic gene delivery. By using a vector that encodes a superparamegnetic metalloprotein to introduce the MRI reporter gene into hosts, we can easily monitor the gene expression in a living organism.

Hui-wen Lin
41925526

Tissue-specific expression of glutathione peroxidase gene in guinea pigs

Glutathione peroxidase (GSH-Px) in guinea pigs was used Northern blot analysis and nuclei run on assay to measure gene expression of GSH-Px activity in major tissues such as liver and kidney in guinea pigs. GSH-Px is a selenocysteine containing enzyme that is important in protecting animals form oxidative injury. Guinea pigs were used on this experiment, due to their very low GSH-Px activity in major tissues. This study present the gene expression of GSH-Px is responsible for the tissues specific regulation of GSH-Px activity in guinea pigs. The result showed that GSH-Px gene in guinea pigs is inactive in any tissues, it has activity transcribed in erythroblasts or erythroid cells on the compare study with mice this may due to the necessity of an antioxidant in the erythrocytes which convey oxygen and susceptible to oxidative stress.

Winnie Yum

Doxycycline-dependent photoactivated gene expression in eukaryotic systems

Abstract:

High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.

Vinodh Narasimhan

42100254

qPCR analysis of gene expression in single cell

Because the smallest metabolically functional unit of a living organism is a single cell,many people are trying to analyze the molecular components in single cells. But the gene expression data averaged over ensembles of cells in various stages or timing may mask important matters.To peer into expression in single cells, Kambara and his colleagues attached cDNA libraries to dyna-beads coated with a polymer. For the first couple of PCR rounds, the fragments are the template, but then the beads sink to the bottom. To allow for the beads to be reused, the qPCR reaction progresses at a lower-than-usual temperature — the polymer on the bead is heat-sensitive.This qPCR analysis for multiple gene expression is accurate enough for various applications and should bring new perspectives to the understanding of complex biological processes.

zai yang Phua
41090714

RNase Protection Assay (RPA) for quntifying Gene Expression Levels

RNase protection assay (RPA) is one of the most common and standard techniques that has been used for analysing the level of gene expression by quantifying mRNA molecules in the cell. This technique provides more sensitivity and reproducibility for measuring mRNA more than the conventional approaches such as Northern blotting. The principle of the technique is based on the hybridization of the target mRNA to a radioactively labelled RNA probe. The hybridized part of the complex becomes protected whereas the unhybridized part of the RNA probe is digested with specific ribonuclease. The hybrid can be resolved by a denaturing gel. Subsequent detection will reveal the appropriate-sized gel band corresponding to the target mRNA. The major advantage of RPA over the other techniques is that the expression of up to 10 or 12 mRNAs can be studied simultaneously in a given sample.

Reference:
http://www.springerlink.com/content/r413643m921186m6/fulltext.pdf

Amjad yousuf
41892954

β-lactamase reporter system for selecting high-producing yeast clones

        In the production of therapeutic proteins in biopharmaceutical industry, it is needed to develop a good screening and selection method for highly-productive clones to lower the production cost. In yeast Pichia pastoris, it has been shown to be a good protein expression system because a new strain with mammalian-type glycosylation was found. However, in P. pastoris, high expression clones are conventionally selected by using different concentrations of antibiotics to screen strains with multiple copies of the expression vector. This method has its limitation due to the limitation in number of colonies screened and the protein expression level is not always correlated with vector copy numbers. Another way of determining the high expression clones is to use GFP as a reporter protein, but the fusion protein is not a good choice for pharmaceutical proteins.

A rapid and simple method to select highly-productive clones is created. This novel expression vector contains two independent expression cassttes. One of these has multiple cloning sites to insert the gene of interest (tested by GFP as a model) controlled by a strong, inducible promoter, AOX1; the other one is consisted of a β-lactamase reporter gene under the control of a weak promoter, YPT1. Clones producing high level of GFP can be selected directly on the plate depending on the intensity of brown color product by β-lactamase. The activity of β-lactamase was found to be proportional to the fluorescence of GFP, which indicated the production level of GFP protein. This reporter system can further be applied to other yeast expression systems such as S. cerevisiae and H. polymorpha.

Reference:β-lactamase reporter system for selecting high-producing yeast clones. BioTechniques 44:477-484 (April 2008)

Yi-Lin (Irene) Cheng
42134932

Promoter swapping, a powerful technique

The ability to control gene expression for many applications, including biomass production using metabolic pathways and functional analysis of cellular processes, has become feasible due to the development of a powerful technique called promoter swapping. This technique which is one type of recombineering involves in the replacement of promoter sequences with engineered promoters using homologous recombination catalysed by phage-derived recombination proteins to alter gene expression. Recombineering was applied firstly in E.coli, and then extended to other organisms including Yersinia, Vibrio, Streptomyces, Mycobacterium, and Pseudomonas.

For marvellous ability of this technique, refer to:

McCleary , William R, 2009. Application of promoter swapping techniques to control expression of chromosomal genes. Appl Microbiol Biotechnol, 84, 641-648.
http://www.springerlink.com/content/63kl5837t483527q/fulltext.html

My Ngoc Nghiem
ID: 42136860

Competitive genomic polymerase chain reaction

Competitive genomic polymerase chain reaction (CGP)

Variation in DNA copy number occurs in many diseases such as Down syndrome as well as in cancer. It is important to detect abnormalities in DNA copy number as associations between DNA aberrations and disease phenotype can be made and critical genes can be located.

Comparative genomic hybridization (CGH) is the standard used for genome wide analysis of DNA copy number. It is based on a two-colour fluorescence in situ hybridization (FISH). Resolution of closely spaced aberrations is not good this making it difficult to assign genomic locus. Results must also be adjusted for biases in the correlation of the heteroscedastic data distribution. Microarray based methods have higher resolution compared to CGH. However, the available arrays have gaps between probes and some regions are not available for analysis.

Competitive genomic PCR is a very useful technique to overcome the limitations. It is performed using restricted genomic DNA ligated to specific adaptors as a template. Different adaptors are added to the test and control samples and the test: reference ratio is determined by quantifying the amplified products fractionated by gel electrophoresis. In the paper, MYCN gene copy alterations in neuroblastoma- derived cells lines are being studied using CGP assay. Gene amplification in each cell line was detected. Detailed high resolution analysis by the CGP assay around the locus revealed new junctions for amplification that were not detected when a commercial array was used. It was also shown that CGP is very sensitive in detecting change in low level DNA copy number. In addition, PCR is an affordable and moderate throughput technique. It can be used to complement the hybridization technique currently used commercially for genomic analysis.

Reference:
Iwao-Koizumi K, Maekawa K, Nakamura Y, Saito S, Kawamoto S, Nakagawara A, Kato. A novel technique for measuring variations in DNA copy- number: competitive genomic polymerase chain reaction. BMC Genomics. 2007; 8: doi: 10.1186/1471- 2164- 8- 206

Loh Mun Jo-anne
(s4184551)

fluorescence in situ hybridization

FISH stands for fluorescence in situ hybridization. It is a cytogenetic technique which is commonly used to probe the presence or absence of a particular DNA sequences on chromosomes. Fluorescent probes only bind to those parts of the chromosome with which they show a high degree of sequence similarity. In this paper, multiple expression domains for SpBrn1/2/4 have been discovered by fluorescent in situ hybridization. Endodermal expression of this gene can be detected in the foregut. SpBrn1/2/4 is also expressed in two distinct ectodermal domains: throughout the stomodeal ectoderm, and within cells scattered throughout the ciliated band. In addition, with this cytogenetic technique, scientists have discovered a unique ectodermal cell type that co-expresses the ParaHox gene SpLox.

More details are available from:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W8W-4VPV5GT-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1043198949&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=be121d4546e01497a6436e71b4659109

Qiushi Chen
41560637

Nanopore Sequencing

“Faster- Cheaper -Easier -More Accurate”

Have you ever thought of protein nanopores, able to read what is there in your DNA?? Surprised, but group of scientist have discovered a novel way of de novo DNA sequencing using protein nanopores. This could be the break through in next generation sequencing approach and could sequence a mammalian genome in approximately not more than a single day in just 1000 dollars. What could be more amazing than this when one could easily find out what is stored in ones genome, in a cost-effective way and much faster than one could imagine. Nanopore sequencing has the potential to sequence nucleotides label free and without the need of amplification. It makes use of a biological nanopore, Staphylococcal alpha-haemolysin protein pore.It is when across the protein pore channel the DNA strand is passed,every nucleotide creates modulation in the ionic current and thus can be detected, as each of the nucelotide base (A,T,G,C) show acharacteristic decrease in current amplitude.It also can distinguish methylated cytosine from the four standard DNA bases, showing its potential in studying epigenetic modifications. At the same time it may also open the possibility for the high-resolution analysis of chromosomal structure variation, and long-range haplotype mapping. This third generation sequencing is currently being developed by Oxford Nanopore Technologies.

To get fascinated more, click here http://www.nature.com/nnano/journal/v4/n4/abs/nnano.2009.12.html


References:

1. James Clarke, Hai-Chen Wu, Lakmal Jayasinghe, Alpesh Patel1, Stuart Reid & Hagan Bayley. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotechnology 4, 265 - 270 (2009)

2. Daniel Branton et al. The potential and challenges of nanopore sequencing. Nature Biotechnology 26, 1146 - 1153 (2008)


Samikshya Biswal
41568400

ChIP-seq technique

ChIP-seq TECHNIQUE
ChIP-Seq is an emerging technique of chromatin immunoprecipitation for the research on gene regulation and epigenetic mechanism. Comparing with conventional ChIP-chip methods, the application of next generation sequencing ChIP-seq that is available for measurement of gene expression through epigenome profiling of DNA- protein interaction, histone modification and nucleosome gives higher resolution with less noise and greater coverage. One current research on gene regulation has defined that tissue-specific activity of enhancers can be accurately predicted by using ChIP-seq.
References
Park, P.J. (2009). ChIP–seq: advantages and challenges of a maturing technology Nature reviews genetics, 10: 669-680.
Visel, A; Matthew, J.; Blow, M.J.; Li Z.; Zhang, T.; Akiyama, J.A.; Holt, A.; Plajzer-Frick I, Shoukry, M.; Wright, C.; Chen, F. et al (2009). Nature, 457: 854-858
Dinh Hoang Lan Chi
Student No: 41614987

Technique blog posts

From this point forward, please use the blog to post a brief description and links to the technique that you will discuss in class.