chromatin conformation capture
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Chromosome conformation capture techniques are a set of molecular biology approaches used to analyze the spatial organization of chromatin and interaction of genomic regions in a cell.
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This document provides information about epigenetics and histone modifications. It defines epigenetics as heritable changes in gene function that do not involve changes to the underlying DNA sequence. It discusses how histone modifications such as acetylation and methylation regulate gene expression by altering chromatin structure and recruiting other proteins. DNA methylation is also described as an important epigenetic modification that typically represses transcription. Several families of enzymes that establish these modifications, such as DNA methyltransferases and histone methyltransferases/acetyltransferases, are outlined.
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Cath
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The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
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The CATH database hierarchically classifies protein domains obtained from protein structures deposited in the Protein Data Bank. Domain identification and classification uses both manual and automated procedures. CATH includes domains from structures determined at 4 angstrom resolution or better that are at least 40 residues long with 70% or more residues having defined side chains. Submitted protein chains are divided into domains, which are then classified in CATH.
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2. There are two main types of chromatin remodeling - covalent histone modification and ATP-dependent chromatin remodeling complexes.
3. ATP-dependent complexes use energy from ATP hydrolysis to move, eject, or restructure nucleosomes, allowing access to DNA.
4. Examples of chromatin remodeling complexes include SWI/SNF, ISWI, CHD, and INO80 families, which have different activities like nucleosome sliding or histone variant exchange.
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Serial Analysis of Gene Expression (SAGE) is a method to quantify gene expression in cells. It involves extracting short sequence tags from mRNA transcripts and concatenating them for efficient sequencing. This allows simultaneous analysis of thousands of transcripts. SAGE provides quantitative gene expression data without prior knowledge of genes and can identify differentially expressed genes between cell types or conditions. While powerful, it requires substantial sequencing and computational analysis of large datasets.
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Repetitive sequences in the eukaryotic genome
Cot curve dispersed repeated DNA or interspersed repeated DNA tandem repeated DNA Long interspersed repeat sequences (LINEs) Short interspersed nuclear elements (SINEs) satellite, minisatellite and microsatellite DNA Variable Number Tandem Repeat (or VNTR)
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After sequencing of the genome has been done, the first thing that comes to mind is "Where are the genes?". Genome annotation is the process of attaching information to the biological sequences. It is an active area of research and it would help scientists a lot to undergo with their wet lab projects once they know the coding parts of a genome.
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Zinc finger nucleases (ZFNs) allow for highly targeted editing of the genome. ZFNs consist of a DNA-binding domain made of zinc finger proteins and a DNA-cleaving domain. The ZFN pair binds to a target site and creates a double-strand break, which the cell repairs through non-homologous end joining or homologous recombination, enabling gene knockouts or targeted changes. ZFNs work in many cell types and animal models, providing a more efficient alternative to traditional transgenic techniques. They have applications in functional genomics, cell line engineering, and animal model generation.
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Cromatin Remodeling
1. Chromatin remodeling is the process by which chromatin structure is dynamically modified to allow access of DNA for processes like transcription.
2. There are two main types of chromatin remodeling - covalent histone modification and ATP-dependent chromatin remodeling complexes.
3. ATP-dependent complexes use energy from ATP hydrolysis to move, eject, or restructure nucleosomes, allowing access to DNA.
4. Examples of chromatin remodeling complexes include SWI/SNF, ISWI, CHD, and INO80 families, which have different activities like nucleosome sliding or histone variant exchange.
Gene silencing
Gene silencing refers to epigenetic processes that regulate genes by switching them off without genetic modification. There are two main types: transcriptional gene silencing modifies chromatin to make genes inaccessible for transcription, while post-transcriptional gene silencing destroys or blocks mRNA to prevent translation into proteins. Both mechanisms protect organisms from transposons and viruses by silencing foreign DNA. Gene silencing is an ancient immune response and also regulates endogenous genes during processes like meiosis.
Shotgun and clone contig method
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
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Ab initio protein structure prediction uses computational methods to predict a protein's 3D structure from its amino acid sequence. It relies on conformational searching to generate structure decoys and selecting native-like models. The key factors for success are an accurate energy function, efficient search methods like molecular dynamics or genetic algorithms, and effective selection of models close to the native structure. Model selection approaches include energy evaluations, compatibility scores, clustering of similar decoys, and identifying the lowest energy conformations.
Serial analysis of gene expression
Serial Analysis of Gene Expression (SAGE) is a method that allows for the quantitative analysis of gene expression patterns in cells or tissues without prior knowledge of gene sequences. It works by extracting short sequence tags (10-14 base pairs) from the 3' end of transcripts and linking them together in long strings that can be cloned and sequenced simultaneously. This allows for many transcripts to be analyzed from a single sequencing reaction and provides quantitative data on gene expression levels based on tag frequencies. SAGE provides an accurate way to discover genes and analyze overall expression patterns without needing to know full mRNA sequences in advance.
Comparative genomic hybridization
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows detection of copy number variations between a test and reference DNA sample without cell culturing. CGH involves labeling and hybridizing test and reference DNA to normal metaphase chromosomes before visualizing differences in fluorescence to identify regions of gains or losses. While CGH was originally used for cancer research, it can also detect chromosomal abnormalities associated with genetic disorders and has improved resolution over traditional cytogenetic methods. The main limitations of CGH are its inability to detect structural aberrations without copy number changes and resolutions above 5-10 megabases.
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Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM
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Title: Exploring Advances in Cytogenetics and Molecular Cytogenetics
Description:
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Key Points:
Introduction to Cytogenetics: Explore the fundamental principles of cytogenetics, its historical significance, and the recent influence of molecular tools, leading to the emergence of molecular cytogenetics.
Importance in Crop Improvement: Uncover the pivotal role of molecular cytogenetics in crop improvement programs, offering insights into the structural and functional organization of genomes within chromosomes.
Karyotyping: Gain a comprehensive understanding of karyotyping, its significance in identifying chromosomal abnormalities, and its applications in studying evolutionary relationships among different taxa.
Chromosome Identification and Sorting: Learn about the techniques involved in the identification and sorting of individual chromosomes, crucial steps in cytogenetics research for various crops.
Chromosome Banding Techniques: Explore different chromosome banding techniques, such as G-Banding and C-Banding, and understand their applications in detecting structural rearrangements.
CHIAS (Chromosome Image Analyzing System): Get insights into the CHIAS software and its role in mapping and identifying chromosomes automatically.
Flow Cytometry: Discover the applications of flow cytometry in detecting and measuring physical and chemical characteristics of cells, with a focus on its relevance in chromosome research.
In Situ Hybridization: Explore the technique of in situ hybridization, particularly the fluorescent variant, and its applications in precise localization of specific DNA segments.
Genomics and Whole Genome Sequencing: Delve into the realm of genomics and whole-genome sequencing, understanding the approaches like BAC to BAC and Whole Genome Shotgun.
Case Study: Uncover a case study involving the identification of a Wheat-Psathyrostachys huashanica ditelosomic addition line, showcasing the practical applications of the discussed techniques.
Conclusion: Summarize the key takeaways from the presentation, emphasizing the role of these techniques in advancing precision breeding and crop improvement.
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- 3. Chromosome conformation capture techniques are a set of molecular biology approaches used to analyze the spatial organization of chromatin and interaction of genomic regions in a cell. 3C 4C 5C Hi-C The Hi-C method allows unbiased, genome- wide identification of chromatin interactions. What Is CCC family ?
- 6. Cells are fixed in 1% final concentration of formaldehyde After crosslinking, the remaining formaldehyde is isolated with an excess of glycine. Then the cells are harvested by centrifugation 1. Cell Culture and Crosslinking of Chromatin
- 7. 2. Cell Lysis and Chromatin Digestion
- 8. Restriction Enzyme Digestion Accessible chromatin is then digested with a type II restriction endonuclease, e.g. HindIII, at 37 C overnight. The HindIII enzyme recognizes the sequence:5’-AAGCTT-3’and cleaves the DNA, leaving a 5’ overhang of: 5’-AGCT-3’. Filling Overhangs The 5’ overhang is filled in by the DNA polymerase I using equimolar amounts of all deoxyribonucleotides
- 9. This cleavage provides a template for labeling the restriction fragments with biotin-14-dCTP The ligation of two completely filled-in HindIII sites forms a NheI site: 5’ -GCTAGC-3’ 3.Biotin marking of DNA ends and blunt end ligation
- 10. The chromatin complexes containing the biotin-labeled ligation products are degraded by incubation with Proteinase K at 65 C. 5.Streptavidin pull-down Molecules with internal biotin incorporation are pulled down with streptavidin coated magnetic beads and modified for deep sequencing. 4.DNA Purification
- 11. 6.Paired-End Adapter Ligation and Library Amplification Ligation of the Illumina Paired-end Adapters is while the Hi-C library is bound to the streptavidin beads. The adsorption of the DNA to the beads increases the efficiency of adapter ligation by decreasing the mobility of the DNA fragments. PCR amplify the library
- 12. Hi-C data visualization and analysis
- 13. Advantages of HI-C 1. High resolution compared to 3C 2. Characterize the structure of the entire genome. 3. It uncovers large blocks of multiple interactions between one chromosome and another. 4. Quantify interactions between all possible pairs of fragments simultaneously. 5. High throughput sequencing . 6. Data produced with hi-c is more comprehensive then other 3-c type methods. 7. Correlated genome architecture with several genomic features like replication timing.
- 14. Cont. 8. Interactions can be detected even over relatively large genomic-distances. 9. Exhibit a high cis-/trans-interaction ratio 10. Hi-C can applied to single cells 11. Detect both known and novel, balanced and unbalanced chromosomal rearrangements from cell lines and human tumour samples 12. Due to high sequence coverage not being required, Hi-C costs significantly less than deep WGS 13. Hi-C does not require dividing cells and can be used on all nucleated cell types
- 15. Disadvantages of HI-C 1. Less resolution then 4C. 2. Workload increase compared to 4C and 5C. 3. Complexity of HI-C library is very high. 4. Hi-C technique cannot distinguish whether interactions are stable and present in some cells, and non-existent in others 5. Background noise is one of the major disadvantage. 6. Evaluation of the random ligation noise in the final library is expensive and time consuming. 7. HI-C has relatively few biases.
- 16. References • https://www.ncbi.nlm.nih.gov/pmc/articles/P MC3874846/ • https://www.researchgate.net/figure/Long- range-chromatin-interactions-revealed-by- HiC-A-Key-steps-of-the- experimental_fig3_273205266 • https://bioconductor.org/help/course- materials/2015/CSAMA2015/lect/L13-hi-c- pekowska.pdf