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.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
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.
DNA Sequencing : Maxam Gilbert and Sanger SequencingVeerendra Nagoria
DNA sequencing is a technique to find out the exact arrangement of Nucleotides to make one strand of DNA. DNA sequencing helps in numerous ways from sequence information to paternity testing, mutation detection etc. Traditionally two approaches were used to solve the problem. First is based of enzymes and Second is based on ddNTPs to sequence the DNA using gel electrophoresis technique.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
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.
DNA Sequencing : Maxam Gilbert and Sanger SequencingVeerendra Nagoria
DNA sequencing is a technique to find out the exact arrangement of Nucleotides to make one strand of DNA. DNA sequencing helps in numerous ways from sequence information to paternity testing, mutation detection etc. Traditionally two approaches were used to solve the problem. First is based of enzymes and Second is based on ddNTPs to sequence the DNA using gel electrophoresis technique.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may cause disease.
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
A chart showing the fate of each part of an early embryo, in a particular blastula stage is called fate maps. It is done because the correct interpretation of gastrulation is impossible without the knowledge of the position which are the presumptive germinal layers (Ectoderm, Mesoderm and Endoderm) occupy in blastula.
Fate mapping is a method used in developmental biology to study the embryonic origin of various adult tissues and structures. The "fate" of each cell or group of cells is mapped onto the embryo, showing which parts of the embryo will develop into which tissue. When carried out at single-cell resolution, this process is called cell lineage tracing. It is also used to trace the development of tumors.
DNA sequencing is the process of determining the sequence of nucleotides (A, T, G, and C) in the DNA. It includes method or technology that is used to determine the order of the four bases: adenine, thymine, guanine and cytosine.
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
Cloning is the process of producing genetically identical individuals of an organism either naturally or artificially.
It is the process of taking genetic information from one living thing and creating identical copies of it. The copied material is called a clone.
Nature has been doing it for millions of years. For example, identical twins have almost identical DNA, and asexual reproduction in some plants and organisms can produce genetically identical offspring.
Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or DNA fragments (molecular cloning).
Bacteriophage- types, structure and morphology of t4 phage, morphogenesisDr. Dinesh C. Sharma
Escherichia virus T4 is a species of bacteriophages that infect Escherichia coli bacteria. It is a member of virus subfamily Tevenvirinae (not to be confused with T-even bacteriophages, which is an alternate name of the species). T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.
Each cell of a multicellular organism contain the same genetic material, but the expression of the gene is different in different type of cell group. On the basis of expression requirement they are grouped in to
Structural Gene- Mostly expressed once in a life
Vital Gene- Involved in of vital biochemical processes such as respiration and need to be expressed all the time
Functional Gene- Genes are not expressed all the time. They are switched on an off at need
The regulation of Gene required in case of functional gene and its explained by Francois Jacob, Jacques Monod and Andre Lwoff (Nobal Prize in 1961)
From studies and predictions such as Dreyer and Bennett's, it shows that the light chains and heavy chains are encoded by separate multigene families on different chromosomes. They are referred to as gene segments and are separated by non-coding regions. The rearrangement and organization of these gene segments during the maturation of B cells produce functional proteins. The entire process of rearrangement and organization of these gene segments is the vital source where our body immune system gets its capabilities to recognize and respond to variety of antigens.
The cells of the B line synthesize immunoglobulins. They are either produced at a membrane (on the surface of the B-lymphocytes) or are secreted (by the plasmocytes)
Theory of preformation,
Epigenetic theory,
Theory of pengenesis,
Recapitulation theory,
Germplasm theory,
Mosaic theory,
Regulated theory,
Gradient theory
Theory of organizers.
Sericulture is the cultivation of silkworms to produce silk. Bombyx mori (the caterpillar of the domesticated silk moth) is the most widely used species of silkworms.
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
1. Sanger sequencing
The chain termination method
By-
Dr. Dinesh C. Sharma
Head, Zoology
K.M. Govt. Girls P. G. College
Badalpur, G.B. Nagar
dr_dineshsharma@hotmail.com
2. DNA sequencing is the process of determining
the sequence of nucleotides (A, T, G, and C) in
the DNA. It includes method or technology that
is used to determine the order of the four bases:
adenine, thymine, guanine and cytosine.
Sequencing an entire genome of an organism
remains a complex task. It requires breaking
the DNA of the genome into many smaller
pieces, sequencing the pieces, and assembling
the sequences into a single long "consensus."
But, new methods developed over the past two
decades make it easier, genome sequencing is
now much faster and less expensive than it was
during the Human Genome Project.
3. 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.
The Sanger method, in mass production form, is the technology
which produced the first human genome in 2001. In the Human
Genome Project, Sanger sequencing was used to determine the
sequences of many relatively small fragments of human DNA.
(These fragments weren't necessarily 900 bp or less, but
researchers were able to "walk" along each fragment using
multiple rounds of Sanger sequencing.) The fragments were
aligned based on overlapping portions to assemble the sequences
of larger regions of DNA and, eventually, entire chromosomes.
Although genomes are now typically sequenced using other
methods that are faster and less expensive, Sanger sequencing is
still in wide use for the sequencing of individual pieces of DNA,
such as fragments used in DNA cloning or generated
through polymerase chain reaction (PCR).
It was first commercialized by Applied Biosystems in 1986
4. Requirement for Sanger sequencing
Sanger sequencing make many copies of a target DNA
region. Its raw material are similar to the requirement of
DNA replication in an organism, or for polymerase chain
reaction (PCR), which copies DNA in vitro.
They include:
• A DNA polymerase enzyme
• A primer, acts as a "starter" for the DNA polymerase
• The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• The template DNA to be sequenced
Sanger sequencing reaction also contains a unique
ingredient:
• Dideoxy nucleotide (dd), or chain-terminating,
versions of all four nucleotides (ddATP, ddTTP, ddCTP,
ddGTP), each labeled with a different color of
dye
5. Dideoxy nucleotides lack a hydroxyl
group on the 3’ carbon of the sugar
ring. In a regular nucleotide, the 3’
hydroxyl group acts as a “hook,"
allowing a new nucleotide to be added
to an existing chain.
Once a dideoxy nucleotide has been
added to the chain, there is no
hydroxyl available and no further
nucleotides can be added.
The chain ends with the dideoxy
nucleotide, which is marked with a
particular color of dye depending on
the base (A, T, C or G) that it carries.
normal
deoxynucleotidetriphosphates
(dNTPs)
modified
di-deoxynucleotidetriphosphates
(ddNTPs),
9. 3”
5”
5”
3”
| | | | | | | | | |
2-Denturation of DNA by heating @ 95O C
3”
5”
5”
3”
Heat Heat
To produce a complimentary strand and the template strand for DNA sequencing
10. 3” T A C G C A T
A 5”
T A T
3”| | |
4-The Primed DNA is then dispersed equally among for vessels
<------- Template strand
<------- Primer
3” T A C G C A T
A 5”
T A T
3”| | |
3-A Primer is then annealed to the 5” end of DNA
<------- Template strand
<------- Primer
11. 5-DNA polymerase is added to all 4 reaction vessels
DNA P DNA P DNA P DNA P
6-Add all four (dATP, dTTP, dGTP, dCTP) to each vessels
A,G,C,
T
A,G,C,
T
A,G,C,
T
A,G,C,
T
12. 7-Modifeid ddNTP are added to reaction vessels
ddAT
P
ddTT
P
ddGT
P
ddTT
P
A T
G C
3” T A T G C A T
A 5”
T A T
3”| | |
<------- Template strand
<------- Primer
13. 3” T A T G C A T
A 5”
T A T
3”| | |
<------- Template strand
<------- Primer
A
T
C
G
DNA
Polymeras
e
ddAT
P
8- The DNA polymerase attaches the dNTP to the template
strand at the primer normally until ddNTP base is pared. As
ddNTP attached the chain termination occur.
14. 3” T A T G C A T
A 5”
T A T
3”| | |
<------- Template strand
<------- PrimerA T
3”
9-Once the ddNTP is based paired , the sequence is
terminated because ddNTP lacks the –OH group at 3’ carbon
ddAT
P
ddTT
P
ddGT
P
ddCT
P
10-As a result of chain termination, DNA fragments of
different length are formed in all four vessels
15. A T G C
Polyacrylamide Gel
Electrophoresis is
used to sequence
DNA
16. A CGT
• DNA migrates form
the –ve pole towards
the +ve pole, due to
–ve charge impaired
by phosphate back
bone
• Smaller (lighter)
DNA fragments
migrate more
rapidly than larger
DNA fragments
• As a result of this
different bands are
observed on plate Small & lighter
Large & Heavy
17. A CGT
The sequence is read form
the bottom of the plate
T
C
A
T
G
G
T
A
T
T
C
A
C
G
G
A
T
A
G
T
C
G
A
18. 5” A G C T G A T A G G C A C T T A T G G T A
C T 3”
T
C
A
T
G
G
T
A
T
T
C
A
C
G
G
A
T
A
G
T
C
G
A
3” T C G A C T A T C C G T G A A T A C C A T
G A 5”
19. Method of Sanger sequencing
• The DNA sample is divided into four separate
sequencing reactions, containing all four of the
standard deoxynucleotides (dNTP, A,C,G,T) and
the DNA polymerase.
• To each reaction is added only one of the four
dideoxynucleotides (ddNTP ddATP, ddTTP,
ddGTP, ddCTP), while the other added
nucleotides are ordinary ones (dN).
• The ddNTP concentration should be
approximately 100-fold higher than that of the
corresponding dNTP (e.g. 0.5mM ddTTP :
0.005mM dTTP) to allow enough fragments to
be produced while still transcribing the
complete sequence (but the concentration of
ddNTP also depends on the desired length of
sequence)
20. • Four separate reactions are needed in this process
to test all four ddNTPs. Following rounds of
template DNA extension from the bound primer,
the resulting DNA fragments are heat denatured
and separated by size using gel electrophoresis.
• In the original publication of 1977, the formation of
base-paired loops of ssDNA was a cause of serious
difficulty in resolving bands at some locations. This
is frequently performed using a denaturing
polyacrylamide-urea gel with each of the four
reactions run in one of four individual lanes (lanes
A, T, G, C). The DNA bands may then be visualized
by autoradiography or UV light and the DNA
sequence can be directly read off the X-ray film or
gel image.
21. • DNA fragments are labelled
with a radioactive or
fluorescent tag on the
primer , in the new DNA
strand with a labeled dNTP,
or with a labeled ddNTP.
• Chain-termination methods
have greatly simplified DNA
sequencing. For example,
chain-termination-based
kits are commercially
available that contain the
reagents needed for
sequencing, pre-aliquoted
and ready to use.
22. Dye-terminator sequencing
utilizes labelling of the chain terminator ddNTPs,
which permits sequencing in a single
reaction, rather than four reactions
as in the labelled-primer method. In dye-
terminator sequencing, each of the four
dideoxynucleotide chain terminators is labelled
with fluorescent dyes, each of which emit light at
different wavelengths.
Owing to its greater expediency and speed, dye-
terminator sequencing is now the mainstay in
automated sequencing.
Its limitations include dye effects due to differences in the incorporation of the
dye-labelled chain terminators into the DNA fragment, resulting in unequal peak
heights and shapes in the electronic DNA sequence trace chromatogram after
capillary electrophoresis. This problem has been addressed with the use of
modified DNA polymerase enzyme systems and dyes that minimize incorporation
variability, as well as methods for eliminating "dye blobs". The dye-terminator
sequencing method, along with automated high-throughput DNA sequence
analyzers, was used for the vast majority of sequencing projects.
23. Automated DNA-sequencing instruments (DNA
sequencers) can sequence up to 384 DNA samples
in a single batch. Batch runs may occur up to 24
times a day. DNA sequencers separate strands by
size (or length) using capillary electrophoresis,
they detect and record dye fluorescence, and
output data as fluorescent peak trace
chromatograms. Sequencing reactions
(thermocycling and labelling), cleanup and re-
suspension of samples in a buffer solution are
performed separately, before loading samples onto
the sequencer. A number of commercial and non-
commercial software packages can trim low-
quality DNA traces automatically. These programs
score the quality of each peak and remove low-
quality base peaks (which are generally located at
the ends of the sequence). The accuracy of such
algorithms is inferior to visual examination by a
human operator, but is adequate for automated
processing of large sequence data sets.
24.
25. Challenges
• Poor quality in the first 15-40 bases of the sequence due
to primer binding and deteriorating quality of sequencing
traces after 700-900 bases. Base calling software such as
Phred typically provides an estimate of quality to aid in
trimming of low-quality regions of sequences.
• In cases where DNA fragments are cloned before
sequencing, the resulting sequence may contain parts of
the cloning vector. In contrast, PCR-based cloning and
next-generation sequencing technologies based on
pyrosequencing often avoid using cloning vectors.
• One-step Sanger sequencing (combined amplification and
sequencing) methods such as Ampliseq and SeqSharp
have been developed that allow rapid sequencing of
target genes without cloning or prior amplification.
• Current methods can directly sequence only relatively
short (300-1000 nucleotides long) DNA fragments in a
single reaction.
• The main obstacle to sequencing DNA fragments above
this size limit is insufficient power of separation for
resolving large DNA fragments that differ in length by only
one nucleotide.
26. Microfluidic Sanger sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip
application for DNA sequencing, in which the
Sanger sequencing steps (thermal cycling, sample
purification, and capillary electrophoresis) are
integrated on a wafer-scale chip using nanoliter-
scale sample volumes.
This technology generates long and accurate
sequence reads, while obviating many of the
significant shortcomings of the conventional
Sanger method (e.g. high consumption of
expensive reagents, reliance on expensive
equipment, personnel-intensive manipulations,
etc.) by integrating and automating the Sanger
sequencing steps.
27. In its modern inception, high-throughput genome
sequencing involves
• fragmenting the genome into small single-stranded
pieces,
• followed by amplification of the fragments by Polymerase
Chain Reaction (PCR).
Adopting the Sanger method, each DNA fragment is
irreversibly terminated with the incorporation of a
fluorescently labeled dideoxy chain-terminating nucleotide,
thereby producing a DNA “ladder” of fragments that each
differ in length by one base and bear a base-specific
fluorescent label at the terminal base.
Amplified base ladders are then separated by Capillary
Array Electrophoresis (CAE) with automated, in situ “finish-
line” detection of the fluorescently labeled ssDNA
fragments, which provides an ordered sequence of the
fragments. These sequence reads are then computer
assembled into overlapping or contiguous sequences
(termed "contigs") which resemble the full genomic
sequence once fully assembled.
28. Applications of microfluidic sequencing technologies
• Single nucleotide polymorphism (SNP) detection,
• Single-strand conformation polymorphism (SSCP)
• Heteroduplex analysis, and
• Short tandem repeat (STR) analysis.
Resolving DNA fragments according to differences in size
and/or conformation is the most critical step in studying
these features of the genome
A single-nucleotide polymorphism (SNP) is a substitution
of a single nucleotide that occurs at a specific position in
the genome, where each variation is present at a level of
more than 1% in the population.
Heteroduplex analysis (HDA) is a method in biochemistry
used to detect point mutations in DNA since 1992.
Heteroduplexes are dsDNA molecules that have one or
more mismatched pairs, on the other hand homoduplexes
are dsDNA which are perfectly paired
29.
30. Next-generation sequencing
The most recent set of DNA sequencing technologies are
collectively referred to as next-generation sequencing.
There are a variety of next-generation sequencing techniques that
use different technologies. However, most share a common set of
features that distinguish them from Sanger sequencing:
• Highly parallel: many sequencing reactions take place at the
same time
• Micro scale: reactions are tiny and many can be done at once on
a chip
• Fast: because reactions are done in parallel, results are ready
much faster
• Low-cost: sequencing a genome is cheaper than with Sanger
sequencing
• Shorter length: reads typically range from 50-700 nucleotides in
length
Conceptually, next-generation sequencing is kind of like running a
very large number of tiny Sanger sequencing reactions in parallel.
This parallelization and small scale, large quantities of DNA can be
sequenced much more quickly and cheaply with next-generation
methods than with Sanger sequencing.
For example, in 2001, the cost of sequencing a human genome was
almost $100 million In 2015, it was just $1245.