DNA Sequencing by sanger's method. Uses dideoxy method of sequencing. Human genome project's data is completely basedon this method, look in the ppt for more information of how Frederick Sanger discovered this process of DNA sequencing and which was one of the biggest breakthrough in the field of biology and life sciences. This discovery won nobel prize in the year 1980.
DNA Sequencing by sanger's method. Uses dideoxy method of sequencing. Human genome project's data is completely basedon this method, look in the ppt for more information of how Frederick Sanger discovered this process of DNA sequencing and which was one of the biggest breakthrough in the field of biology and life sciences. This discovery won nobel prize in the year 1980.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
Gene Sequencing, a tool to analyze the exact order of nucleotide sequence in the DNA -Deoxyribonucleic Acid.
Focuses on Two methods:
a. Maxam-Gilbert (Chemical Degradation) Method
b. Sanger's Method (Dideoxy Chain termination Method)
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
DNA consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment.[5Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.
The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal, plant, and microbial species.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate.Whole Genome Sequencing
•Allows doctors to closely analyze a patient's genes for mutations and health indicators.
•Can detect intellectual disabilities and developmental delays.
•WGS is currently available at Yale for patients in the NICU and PICU.
•Involves Genetics.Sequencing may be utilized to determine the order of nucleotides in small targeted genomic regions or entire genomes. Illumina sequencing enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome, or epigenome of any organism.The spectrum of analysis of NGS can extend from a small number of genes to an entire genome, depending on the goal. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) provide the sequence of DNA bases across the genome and exome, respectively.Capillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. sanger and Maxam-Gilbert sequencing technologies were classified
Herbal Drug Technology
Herbs as Raw Materials: Definition of herb, herbal medicine, herbal medicinal product and herbal drug preparation, source of herbs, selection, identification and authentication of herbal materials, processing of herbal raw material.
Herbal Excipients : Herbal Excipients – Significance of substances of natural origin as excipients, – colorants, sweeteners, binders, diluents, viscosity builders, dis-integrants, flavors & perfumes.
Herbal Formulations: Stages involved in herbal formulations, Orthodox formulations and methods of delivery of herbal extracts, Novel formulations of herbal extracts.
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
Gene Sequencing, a tool to analyze the exact order of nucleotide sequence in the DNA -Deoxyribonucleic Acid.
Focuses on Two methods:
a. Maxam-Gilbert (Chemical Degradation) Method
b. Sanger's Method (Dideoxy Chain termination Method)
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
DNA consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment.[5Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.
The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal, plant, and microbial species.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate.Whole Genome Sequencing
•Allows doctors to closely analyze a patient's genes for mutations and health indicators.
•Can detect intellectual disabilities and developmental delays.
•WGS is currently available at Yale for patients in the NICU and PICU.
•Involves Genetics.Sequencing may be utilized to determine the order of nucleotides in small targeted genomic regions or entire genomes. Illumina sequencing enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome, or epigenome of any organism.The spectrum of analysis of NGS can extend from a small number of genes to an entire genome, depending on the goal. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) provide the sequence of DNA bases across the genome and exome, respectively.Capillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. sanger and Maxam-Gilbert sequencing technologies were classified
Herbal Drug Technology
Herbs as Raw Materials: Definition of herb, herbal medicine, herbal medicinal product and herbal drug preparation, source of herbs, selection, identification and authentication of herbal materials, processing of herbal raw material.
Herbal Excipients : Herbal Excipients – Significance of substances of natural origin as excipients, – colorants, sweeteners, binders, diluents, viscosity builders, dis-integrants, flavors & perfumes.
Herbal Formulations: Stages involved in herbal formulations, Orthodox formulations and methods of delivery of herbal extracts, Novel formulations of herbal extracts.
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
Metabolomics-Introduction, metabolism, intermediary metabolism, metabolic pathways, metabolites, metabolome, metabolic turnover, techniques used in metabolomics, metabolite profiling methods, data analysis, metabolomic resources, role of metabolomics in system biology.
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
Genome projects
Definition of genome, history of genome projects, whole genome sequencing, Maxam Gilbert sequencing, sanger sequencing, explanation on the first sequenced organisms (Bacteriophage, bacteria, archaeon, virus, bakers yeast, nematode.
Model organism-Arabidopsis thaliana, Mus musculus, Oryza sativa, Pan troglodyte etc.
Human genome project, milestones and significance.
Epigenetics studies stably heritable traits that cannot be explained by changes in DNA sequence.
Covalent modifications in chromatin
DNA- DNA methylation (CpG); hydroxymethylation
Histone - lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation
Epigenetic mechanisms:
Modified histones as post translational modification
DNA methylation – 5mC the 5th base, methyl transferases; genetic imprinting.
Epigenomics: complete set of epigenetic modifications on the genetic material of a cell.
Specific epigenetic regulation
RNA interference
X inactivation (Lyonization)
Genomic imprinting
Epigenetics in development and diseases.
Comparative genomics: Genomic features are compared, evolutionary relationship
The major principle of comparative genomics is that common features of two organisms will often be encoded within the DNA that is evolutionarily conserved between them. orthologous sequences,
Started as soon as the whole genomes of two organisms became available (that is, the genomes of the bacteria Haemophilus influenzae and Mycoplasma genitalium) in 1995, comparative genomics is now a standard component of the analysis of every new genome sequence. comparative genomics studies of small model organisms (for example the model Caenorhabditis elegans and closely related Caenorhabditis briggsae) are of great importance to advance our understanding of general mechanisms of evolution
Computational tools for analyzing sequences and complete genomes. Application of comparative genomics in agriculture and medicine.
Mapping and sequencing genomes: Genetic and physical mapping, Sequencing genomes different strategies, High-throughput sequencing, next-generation sequencing technologies, comparative genomics, population genomics, epigenetics, Human genome project, pharmacogenomics, genomic medicine, applications of genomics to improve public health.
Disorders of liver and kidney, Nitrogen metabolism.pdfshinycthomas
Disorders of liver and kidney – Jaundice, fatty liver, normal and abnormal functions of liver and kidney. Inulin and urea clearance.
Abnormalities of nitrogen metabolism
Lipid metabolism and its disorders.pdfshinycthomas
Disorders of Lipids – Plasma lipoproteins, cholesterol, triglycerides and phospholipids in health and disease, hyperlipidemia, hyperlipoproteinemia, Gaucher’s disease, Tay-Sach’s and Niemann-Pick disease, ketone bodies.
a) Definition, classification, structure, stereochemistry and reactions of amino acids;
b) Classification of proteins on the basis of solubility and shape, structure, and biological functions. Primary structure - determination of amino acid sequences of proteins, the peptide bond, Ramachandran plot.
c) Secondary structure - weak interactions involved - alpha helix and beta sheet and beta turns structure, Pauling and Corey model for fibrous proteins, Collagen triple helix, and super secondary structures - helix-loop-helix.
d) Tertiary structure - alpha and beta domains. Quaternary structure - structure of haemoglobin, Solid state synthesis of peptides, Protein-Protein interactions, Concept of chaperones.
Nucleic acid-DNA and RNA
Gene-part of DNA
Functions of DNA
RNA-Functions, different types of RNA-Ribosomal RNAs (rRNAs), Messenger RNAs (mRNAs), Transfer RNAs (tRNAs)-Other RNA-Small nuclear RNA (snRNA), Micro RNA (miRNA), Small interfering RNA (siRNA), Heterogenous RNA (hnRNA).
Nucleic acid-nucleotides-nucleoside
Components of nucleotide-a nitrogenous (nitrogen-containing) base (pyrimidine and purine), (2) a pentose, and (3) a phosphate
Structure of pentose sugar, and 5 major bases (cytosine, thymine, uracil-pyrimidine bases and adenine, guanine-purine bases).
Deoxyribonucleotides and Ribo nucleotides-Molecular structure of deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP) and Adenosine monophosphate (AMP), Guanosine monophosphate (GMP), Cytosine monophosphate (CMP) and Uridine monophosphate (UMP), Watson crick base pairing, Hoogsteen base pairing,
Helical structure-Heterocylic N -Glycosides, Syn and Anti Conformers, detailed structure of single strand and double stranded DNA.
DNA Nucleotides and Tautomeric Form-Tautomers of Adenine, Cytosine, Guanine, and Thymine
Template strand, non coding strand and coding strand
Hydrogen bonds, phosphodiester linkage, hydrolysis of DNA and RNA.
Different forms of DNA-A, B, and Z forms.
Palindrome sequence, Linear DNA, Cruciform DNA, H DNA (Triplex DNA), Denaturation of DNA, Hyperchromicity, Tm, Renaturation of DNA, Tertiary structure of DNA, Difference of DNA and RNA, RNA structural elements, primary. secondary and tertiary structure of RNA. Detailed structure and functions of tRNA, mRNA, rRNA, miRNA, siRNA, hn RNA, snRNA.
Nucleic acid hybridization, C0t analysis, Buoyant density of DNA, Isopycnic centrifugation.
Lipids-Introduction, properties and functions.
Classification-Simple lipids, complex lipids and derived lipids.
Lipids contain fatty acid and alcohol.
Saturated and Unsaturated fatty acids. Nomenclature of fatty acids, Cis-trans isomerism, essential fatty acids
Simple lipids-Fats, waxes
Compound lipids-Structure, function with examples of Phospholipids, Glycolipids, sulpholipids and lipoproteins.
Derived lipids: Structure, types, and functions of steroids, terpenes and carotenoids.
Lipoproteins-classified into chylomicrons, very low-density lipoproteins (VLDL), low density lipoproteins (LDL) and high-density lipoproteins (HDL) and their function.
Eicosanoids-prostanoids, leukotrienes (LTs), and lipoxins (LXs).
Functions of Eicosanoids
Lipids, micelles and liposomes.
Vitamins-Introduction, Water soluble and fat soluble vitamins.
Water soluble vitamins-B complex vitamins: thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6 (pyridoxine), folate (folic acid), vitamin B12, biotin and pantothenic acid-their source, structure, properties, metabolism, physiological significance, deficiency disease and human requirements.
Fat soluble vitamins: Fat soluble vitamins, Vitamin A, D, E and K and their their source, structure, properties, metabolism, physiological significance, deficiency disease and human requirements.
Vitamin A-Carotene in plants-α-carotenes, β-carotenes and γ-carotenes, 3 forms of vitamin A-Retinol, Retinal, Retinoic acid.
Vitamin D3-cholecalciferol,
Vitamin E -Tocopherol, Vitamin K-Phylloquinone or Anti hemorrhagic Vitamin or Coagulation Vitamin
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Predicting property prices with machine learning algorithms.pdf
Sanger-Shortgun sequencing.pdf
1. Sanger Sequencing: Introduction, Principle, and Protocol
What Is Sanger Sequencing?
Sanger sequencing, also known as the “chain termination method,” was
developed by the English biochemist Frederick Sanger and his
colleagues in 1977.
• This method is designed for determining the sequence of nucleotide
bases in a piece of DNA (commonly less than 1,000 bp in length).
• Sanger sequencing with 99.99% base accuracy is considered the
“gold standard” for validating DNA sequences, including those
already sequenced through next-generation sequencing (NGS).
Dr. Shiny C Thomas, Department of Biosciences, ADBU
2. • Sanger sequencing was used in the Human Genome Project to determine the
sequences of relatively small fragments of human DNA (900 bp or less).
• These fragments were used to assemble larger DNA fragments and,
eventually, entire chromosomes.
Sanger Sequencing VS NGS
The development of NGS technologies has accelerated genomics research.
• NGS can simultaneously sequence more than 100 genes and whole genomes
with low-input DNA.
• Sanger sequencing remains widely used in the sequencing field as it offers
several prominent advantages: (i) cost-efficiency for sequencing single genes
and (ii) 99.99% accuracy, especially suitable for verification sequencing for
site-directed mutagenesis or cloned inserts.
3. How Does Sanger Sequencing Work?
• In Sanger sequencing, a DNA primer complementary to the template DNA
(the DNA to be sequenced) is used to be a starting point for DNA synthesis. In
the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C, and
T), the polymerase extends the primer by adding the complementary dNTP
to the template DNA strand.
• To determine which nucleotide is incorporated into the chain of nucleotides,
four dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and
ddTTP) labeled with a distinct fluorescent dye are used to terminate the
synthesis reaction.
4. • Compared to dNTPs, ddNTPs has an oxygen atom removed from the
ribonucleotide, hence cannot form a link with the next nucleotide.
• Following synthesis, the reaction products are loaded into four lanes of a single
gel depending on the diverse chain-terminating nucleotide and subjected to
gel electrophoresis.
• According to their sizes, the sequence of the DNA is thus determined.
5.
6. Sanger Sequencing Steps
The Sanger sequencing method consists of 6 steps:
(1) The double-stranded DNA (dsDNA) is denatured into two single-stranded
DNA (ssDNA).
(2) A primer that corresponds to one end of the sequence is attached.
(3) Four polymerase solutions with four types of dNTPs but only one type of
ddNTP are added.
(4) The DNA synthesis reaction initiates and the chain extends until a
termination nucleotide is randomly incorporated.
(5) The resulting DNA fragments are denatured into ssDNA.
(6) The denatured fragments are separated by gel electrophoresis and the
sequence is determined.
7.
8. Shotgun Sequencing
• High throughput sequencing method that employs
automated sequencing of random DNA fragments
• Automated DNA sequencing yields sequences of 500 to
1000 bp in length
• To determine longer sequences you obtain fragmentary
sequences and then join them together by overlapping
• Overlapping is an alignment problem, but different
from those we have discussed up to now
9.
10.
11.
12. Multiple Sequence Alignment
• A general method to align and compare more than 2
sequences
• Typically done as a hierarchical clustering/alignment
process where you match the two most similar sequences
and then use the combined consensus sequence to
identify the next closest sequence with which to align