SSH Analysis
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Subtractive hybridization (SSH) is a technique for identifying differentially expressed genes between two populations of nucleic acids. It involves two hybridizations - in the first, an excess of driver cDNA is added to normalize the tester cDNA. In the second hybridization, the normalized cDNA is mixed with more driver cDNA to selectively amplify sequences present in the tester but not driver (E-hybrids). Only the E-hybrids can be exponentially amplified due to their different adapter sequences, allowing isolation of differentially expressed genes between populations. SSH is commonly used for plant and cancer research and to study various diseases.
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1. PCR cloning allows amplification of DNA fragments and vectors without restriction enzymes, requiring only small amounts of DNA.
2. PCR has numerous applications including gene cloning, paternity testing, and mutation detection for inherited diseases.
3. Key aspects of PCR include its three main stages - denaturation, annealing of primers, and extension. Proofreading enzymes play an important role by identifying and removing mismatched bases to ensure high fidelity DNA replication.
RNA Sequencing Research
Single cell RNA-seq was performed on 18 mouse bone marrow dendritic cells. 982 genes were found to be differentially expressed between two cells, while the majority of genes showed similar expression levels. Future work will analyze the functions of differentially expressed genes to better understand heterogeneity between cells and potential roles in disease.
Random amplified polymorphic DNA-RAPD.pptx
Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.
Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.
Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.
5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.
Lectut btn-202-ppt-l28. variants of pcr-ii
Reverse transcriptase PCR (RT-PCR) is used to amplify cDNA copies of RNA. It involves reverse transcribing RNA to cDNA then amplifying the cDNA with PCR. RT-PCR can be used to study gene expression and diagnose genetic diseases. Variations include band-stab PCR which reamplifies low yield fragments, degenerate PCR which uses mixed primers for related gene families, and anchored PCR which attaches a known sequence to amplify unknown 5' sequences. Real-time PCR monitors fluorescence during amplification to quantify templates in each cycle, allowing visualization of reactions in real-time. It is commonly used to measure changes in gene expression.
Ashg poster sp_compressed
This document analyzes PCR duplicates and library diversity in RNA-seq. It finds that under normal assay conditions, different RNA-seq assays give different apparent numbers of duplicates on the same samples, due to differences in transcript complexity rather than PCR artifacts. Using molecular barcoding, there are lower levels of true PCR duplicates in standard RNA-seq preps. However, reducing input amounts below 10ng leads to dramatic increases in duplicates. Even with low input amounts that produce many duplicates, duplicate reads still accurately measure gene expression levels and are amplified uniformly rather than with bias.
Chapter8 igenetics
The document provides an overview of recombinant DNA technology and cloning techniques. It discusses:
1) The general steps to clone DNA - isolating DNA from an organism, cutting it with restriction enzymes to create recombinant DNA, and introducing the DNA into a host.
2) Types of cloning vectors like plasmids, artificial chromosomes, and viruses that are used to clone DNA fragments. Genomic and cDNA libraries containing clones of all DNA sequences are also described.
3) Techniques for identifying recombinant clones like hybridization probes, complementation of mutations, restriction mapping, and sequencing.
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Loop mediated isothermal amplification by dr.pavulraj.s
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Characteristics of Loop Mediated Isothermal Amplification Technique
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Deep Software Variability and Frictionless Reproducibility
Deep Software Variability and Frictionless ReproducibilityUniversity of Rennes, INSA Rennes, Inria/IRISA, CNRS
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
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Exposé invité Journées Nationales du GDR GPL 2024
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SSH Analysis
- 1. SUBSTRACTIVE HYBRIDIZATION (SSH ANALYSIS) Submitted By, LOGESWARAN KA P20BIT1013 I-MSC. BIOTECHNOLOGY PERIYAR UNIVERSITY.
- 2. CONTENTS INTRODUCTION PRINCIPLE WORKFLOW OF PCR SUPPRESSION EFFECT HYBRIDIZATIONS (FIRST & SECOND) CONCLUSIVE EVENTS PROS AND CONS CURRENT RESEARCH BIBLIOGRAPHY
- 3. INTRODUCTION PCR Based cDNA Substraction Procedure. Pioneers – Luda Diatchenko, Sergey Lukyanov And His Colleagues In 1996. Very Effective In Tissue Specific cDNA synthesis And Functional Sequences Identification. Mainly Based On PCR Suppression Effect (Panhandle Like Loop Structure Formation) Caused By Long ITRs (Inverted Terminal Repeats). Undesired DNA Fragments Can Be Removed From The Mixture Of Target DNA Samples. One Round Of SSH Analysis Results In 1000 Fold Differentially Expressed cDNA Synthesis. Removed cDNA Mixture Can Be Used As Hybridization Probe For Screening rDNA Libraries.
- 4. PRINCIPLE Driver And Tester cDNA Samples Was Prepared. Tester Contains Target cDNA And Driver Contains No Or Low Amount Of Target cDNA. Both Were Exposed To 4-Base Blunt End Cutting Restriction Enzymes And Digested. The Resulting Tester Sample Divided Into 2 Portions (A&B) And Ligated With 2 Different Adapters (A&B) At 5’End And 2 Populations Of Tester Samples Were Prepared. The Adapters Designed Without PO4-Groups For Covalent Binding Of Longer Strand Of Both Adapters (A&B) To 5’End Of cDNA. SSH Analysis Contains 2 Hybridizations. First Hybridization. Second Hybridization.
- 5. Workflow Of PCR Suppression Effect
- 6. HYBRIDIZATIONS FIRST HYBRIDIZATION Excess Driver Sample Added To Both Tester Samples A&B. Heat Denatured And Allowed To Anneal. Tester A Becomes Normalised Due To Homohybrid cDNA Production. So, It Is Enriched With cDNA For Differential Expression . Normalisation Means Concentrations Of High and Low Abundance Of cDNA In A Samples Becomes Roughly Equal. Tester B Is Faster For More Abundant Compounds Due To Second Order Kinetics. SECOND HYBRIDIZATION Mixing Of 2 samples From First hybridization. New Hybrids B, C, D, And E Were Produced By Reassociation. Another Portion Of Driver Is Added And It Enriches E- Hybrids For Differential Expression. E-Hybrids Has Different Adapter Sequences At Their 5’End. One Is From Tester Sample A And Another One Is From Tester Sample B. These 2 Adapter sequences Ensures Proper Amplification Of E-Hybrids In PCR.
- 7. CONCLUSIVE EVENTS Exponential Growth Only Takes Place In E-Hybrids. E-Hybrids Has Different Adapter sequences At Their 5’End. B-Hybrids Cannot Be Served As Template For Exponential Growth. Because, They Have Long ITRs At The End And Results In PCR Suppression Effect (Formation Of Panhandle Like Structure) Due To Intramolecular Annealing. A-Hybrids And D-Hybrids Do Not Have Primer Binding Sites. C-Hybrids Can Be Amplified At Linear Rate. So, Only The E-Hybrids With Different Adapter Sequences At The End Can Be Amplified Exponentially. In This Method, Undesirable Sequences Can Be Removed And Selective Amplification Of cDNA Can Be Achieved.
- 8. PROS AND CONS PROS Characterization Of Two Populations Of Nucleic Acids. Substraction Of Plant And Bacterial Genomes. Generation And Cloning Of Tissue Specific cDNA. Isolation Of Differentially Expressed Genes. cDNA And Genomic DNA Library Construction. Detection Of DNA And RNA Differences Between Different Genomes, Cells And Organisms. CONS During RNA Comparison, Few Micrograms Of Poly(A)+ RNA From 2 Cell Populations Is Needed. It Is Difficult To Obtain. Appearance Of False Positive DNA During DNA Studies Is A Major Drawback. Mirror Orientation Selection (MOS) Is The Solution.
- 9. CURRENT RESEARCH Plant Genome Research To Fight Extreme Environmental Conditions. Different Cancers Treatments. Different Animal Diseases Treatments. Different Human Diseases Treatments. Different Bacterial Diseases Treatments. Different Fungal Diseases Treatments. Different Viral Diseases Treatments.
- 10. BIBLIOGRAPHY https://www.ncbi.nlm.nih.gov/pmc/articles/PMC39182/ (Pioneers Research Article) https://pubmed.ncbi.nlm.nih.gov/14970460/ https://www.sciencedirect.com/topics/medicine-and- dentistry/suppression-subtractive-hybridization http://evrogen.com/technologies/SSH.shtml http://what-when-how.com/molecular-biology/subtractive- hybridization-molecular-biology/ https://www.ahajournals.org/doi/full/10.1161/01.STR.32.4.10 20 (Case Study - Using SSH Analysis For Differential Gene Expression In Strokes)
- 11. THANK YOU