DNA sequencing involves determining the order of nucleotides in DNA. There are two main methods: the Maxam-Gilbert method which uses chemical reactions to modify specific bases, and the Sanger method which uses DNA polymerase and a mixture of normal and terminated nucleotides to generate fragments of different lengths. These fragments are then separated by gel electrophoresis to determine the DNA sequence.
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.
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.
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
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
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
One of the most developed cities of India, the city of Chennai is the capital of Tamilnadu and many people from different parts of India come here to earn their bread and butter. Being a metropolitan, the city is filled with towering building and beaches but the sad part as with almost every Indian city
Navigating Challenges: Mental Health, Legislation, and the Prison System in B...Guillermo Rivera
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R3 Stem Cells and Kidney Repair A New Horizon in Nephrology.pptxR3 Stem Cell
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India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
Explore our infographic on 'Essential Metrics for Palliative Care Management' which highlights key performance indicators crucial for enhancing the quality and efficiency of palliative care services.
This visual guide breaks down important metrics across four categories: Patient-Centered Metrics, Care Efficiency Metrics, Quality of Life Metrics, and Staff Metrics. Each section is designed to help healthcare professionals monitor and improve care delivery for patients facing serious illnesses. Understand how to implement these metrics in your palliative care practices for better outcomes and higher satisfaction levels.
3. DNA sequencing
Determination of nucleotide sequence
Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
They depend on the production of a mixture of
oligonucleotides labeled either radioactively or
fluorescein, with one common end and differing in
length by a single nucleotide at the other end
This mixture of oligonucleotides is separated by high
resolution electrophoresis on polyacrilamide gels
and the position of the bands determined
4. Maxam and Gilbert Method
The single stranded DNA fragment to be sequenced is end-
labeled by treatment with alkaline phosphatase to remove the
5’phosphate
It is then followed by reaction with P-labeled ATP in the
presence of polynucleotide kinase, which attaches P labeled to
the 5’terminal
The labeled DNA fragment is then divided into four aliquots,
each of which is treated with a reagent which modifies a
specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue
2. Aliquot B + formic acid, which modifies adenine and guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for
cytosine
The four are incubated with piperidine which cleaves the sugar
phosphate backbone of DNA next to the residue that has been
modified
5. Frederick Sanger
• Discovered DNA sequencing by chain
termination method
• Nobel Prize 1 (1958)
– Complete amino acid
sequence of insulin
• Nobel Prize 2 (1980)
– For DNA sequencing
6. Sanger Method
DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
Requires a primer, DNA polymerase, a template, a mixture
of nucleotides, and detection system
Incorporation of dideoxynucleotides into growing strand
terminates synthesis
Synthesized strand sizes are determined for each
dideoxynucleotide rxn by using gel or capillary
electrophoresis
8. Dideoxy nucleotides
• Incorporation of a dideoxynucleotide to
growing DNA strand terminates its further
extension
• Are added in small proportion
– dATP ddATP
– dGTP ddGTP
– dCTP ddCTP
– dTTP ddTTP
15. DNA Sequencing – vectors
+ =
DNA
Shake
DNA fragments
Vector
Circular genome
(bacterium, plasmid)
Known
location
(restriction
site)
16. Different types of vectors
VECTOR Size of insert
Plasmid
2,000-10,000
Can control the
size
Cosmid 40,000
BAC (Bacterial Artificial
Chromosome)
70,000-300,000
YAC (Yeast Artificial
Chromosome)
> 300,000
Not used much
recently
17. DNA Sequencing – gel
electrophoresis
1. Start at primer
(restriction site)
2. Grow DNA chain
3. Include
dideoxynucleoside
(modified a, c, g, t)
4. Stops reaction at all
possible points
5. Separate products with
length, using gel
electrophoresis
20. Extension Chemistry
• Polymerase
– Sequenase
– Thermostable (Cycle Sequencing)
• Terminators
– Dye labels (“Big Dye”)
• spectrally different, high fluorescence
– ddA,C,G,T with primer labels
21. Separation
• Gel Electrophoresis
• Capillary Electrophoresis
– suited to automation
• rapid (2 hrs vs 12 hrs)
• re-usable
• simple temperature control
• 96 well format
