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)
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
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.
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.
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
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.
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 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
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.
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A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
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Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
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2. INTRODUCTION
• DNA is the blueprint of life consisting of chemical building
blocks called nucleotides.
• These building blocks are made of three parts:
phosphate, sugar group, and one of the four types of
nitrogen bases viz adenine (a), thymine (t), guanine (g), or
cytosine (c).
• The order or sequence of these bases determines
what biological instructions are contained in a strand
of dna.
3. • DNA is a double helix
• Two strands run anti parallel to each other
• Paired together by hydrogen bonds
• Highly specific nature of this type of chemical pairing
• A always pairs with base T, and likewise C with G
• Therefore, if the sequence of the bases on one strand of a
DNA double helix is known, it is simple to figure out the
sequence of bases on the other strand.
4. Landmarks in DNA Sequencing
• 1953 Discovery of the structure of the DNA double
helix.
• 1972 Development of recombinant DNA technology.
• 1977 The first complete genome of bacteriophage uX174
sequenced.
• 1977 Allan Maxam and Walter Gilbert publish ‘‘DNA
sequencing by chemical degradation.
• 2001 A draft sequence of the human genome published.
5.
6. What is Gene Sequencing
• DNA sequencing is the process of determining the exact
order of nucleotides within a DNA molecule. This method is
used to determine the order of the four bases—adenine (A),
guanine (G), cytosine (CY), and thymine (T) in a strand of
DNA.
7.
8. Maxam–Gilbert Method
• Allan Maxam and Walter Gilbert developed a method
for sequencing single-stranded DNA by a two-step
catalytic process involving piperidine and two
chemicals that selectively attack purines and
pyrimidines .
9. • This is the best method offered for sequencing of small
oligonucleotides.
• Maxam–Gilbert sequencing is a method of DNA
sequencing
developed by Allan Maxam and Walter Gilbert in 1976.
• Maxam–Gilbert sequencing requires radioactive labeling
at
one 5′ end of the DNA fragment to be sequenced.
• The Maxam and Gilbert chemical sequencing method is
based on the ability of different chemical to specifically
modify bases within the DNA molecule.
• The chemical specificity of the first reactions are as
follows:
G: DMS
G+A: Formic acid
T+C: Hydrazine
C: In the presence of NaCl, only C reacts with hydrazine
10. Procedure (STEPS)
• Radioactive labelling of one end (5' end or 3’ end) of the
DNA fragment to be sequenced by a kinase reaction using
32P.
• Cut the DNA fragment with specific restriction enzyme,
resulting in two unequal DNA fragments
• Denature the double-stranded DNA to single-stranded
DNA by increasing temperature.
• Cleave the DNA strand at specific positions using
chemical reactions.
11. We can use one of the two chemicals followed by
addition of piperdine. Dimethyl sulphate (DMS)
selectively attacks purine (A and G), while hydrazine
selectively attacks pyrimidines (C and T). This is called
modification step.
• Chemical treatment generates breaks at the four
nucleotide bases in the four reaction mixtures (G,
A+G, C, and C+ T).
12.
13. • Fragments are subjected to electrophoresis in high-
resolution acrylamide gels for size-based separation.
• To visualize the fragments, the gel is exposed to X-
ray film for autoradiography, which yields a series of
dark bands, each corresponding to a radiolabeled
DNA fragment, from which the nucleotide sequence
may be inferred.
• In the gel, the fragments are ordered by size and,
thus, we can deduce the sequence of the DNA
molecule.
14.
15. Sanger and Coulson Method
Dideoxynucleotide Chain
Termination Method
• It is an enzymatic method.
• developed by Frederick Sanger and his colleagues in
1977.
• Sanger won the Nobel Prize in Chemistry in 1980 for
development of this technique.
• A powerful technique for sequencing.
• Utilizes single stranded DNA as a template.
16. • Also called dideoxynucleotide termination method.
• Requirements:
• A primer with free 3’-OH ends to start DNA
synthesis
• DNA Polymerase
• dNTPs
17.
18.
19. Feature of ddNTPs
• If any of the ddNTPs binds, the chain elongation is
terminated.
• Because ddNTPs don’t have free 3’-OH end which is
required for chain elongation.
• Therefore, no phosphodiester bond will be formed.
20.
21. Procedure
• Four reaction tubes are labelled with A,T,G and C
each containing -
• Single stranded DNA template – obtained by NaOH
hydrolysis
• 5’-radiolabelled DNA primer and,
• All four radiolabelled dNTPs (dATP, dGTP, dCTP
and dTTP)
22.
23. • A small amount of:
• ddATP is added to tube 1,
• ddGTP to tube 2,
• ddCTP to tube 3 and,
• ddTTP to tube 4.
• Conentration of ddNTPs should be maintained to
about 1% of the concentration of dNTPS
24. Procedure Contd…
• DNA polymerase is added to each tube.
• DNA synthesis starts and chain elongates.
• In each tube ddNTP is randomly incorporated and
fragments are terminated.
• The length of fragments depend on the position of
incorporation of ddNTPs.
25. • After completion of reaction, the fragments of each
tubes are separated by electrophoresis in four
different lanes of high resolution Polyacrylamide
Gel.
• Gel is then dried and autoradiography is done.
• DNA sequence is obtained by reading the bands on
autoradiogram of four lanes.
• (from bottom to top of gel)
26.
27.
28. Significance of DNA Sequencing
• Information obtained by DNA sequencing makes it
possible to understand or alter the function of genes.
• DNA sequence analysis demonstrates regulatory
regions that control gene expression and genetic “hot
spots” particularly susceptible to mutation.
• Comparison of DNA sequences shows evolutionary
relationships that provide a framework for definite
classification of microorganisms including viruses.
29. • Comparison of DNA sequences facilitates identification
of conserved regions, which are useful for development
of specific hybridization probes to detect microorganisms
including viruses in clinical samples.
• DNA sequencing has become sufficiently fast and
inexpensive to allow laboratory determination of
microbial sequences for identification of microbes.
Sequencing of the 16S ribosomal subunit can be used to
identify specific bacteria. Sequencing of viruses can be
used to identify the virus and distinguish different strains.
• DNA sequencing shows gene structure that helps research
workers to find out the structure of gene products.