This document discusses the history and various methods of DNA sequencing. It begins with a brief overview of DNA sequencing and its uses. It then outlines some of the major developments in DNA sequencing techniques, including the earliest RNA sequencing in 1972, Sanger sequencing in 1977, and the first complete genome of Haemophilus influenzae in 1995. The document proceeds to provide more detailed explanations of several DNA sequencing methods, such as Sanger sequencing, pyrosequencing, shotgun sequencing, Illumina sequencing, and SOLiD sequencing.
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.
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.
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.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Synopsis
Introduction
Some Facts
Types of SNPs
SNPs act as gene markers
Methods of Detection
Techniques to detect SNPs
Allelic Specific Cleavage
Differential Hybridization
Single Base Extension or minisequencing
Alternate Methods for Detecting SNPs
Mass Spectrometry
Microchips
SIGNIFICANCE OF SNPs
HAPLOTYPE
ADVANTAGES
Are SNP data available to the public?
Some important SNP database Resources
CONCLUSION
References
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
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.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
https://www.patreon.com/biotechlive
SUPPORT EDUCATION... SUPPORT US
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Synopsis
Introduction
Some Facts
Types of SNPs
SNPs act as gene markers
Methods of Detection
Techniques to detect SNPs
Allelic Specific Cleavage
Differential Hybridization
Single Base Extension or minisequencing
Alternate Methods for Detecting SNPs
Mass Spectrometry
Microchips
SIGNIFICANCE OF SNPs
HAPLOTYPE
ADVANTAGES
Are SNP data available to the public?
Some important SNP database Resources
CONCLUSION
References
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
Detailed explanation about gene sequencing methods
Sequencing the gene is an important step toward understanding the gene.
A gene sequence contains some clues about where genes are.
Gene sequencing give us understanding how the genome as a whole works-how genes work together to direct the growth, development and maintenance of an entire organism.
It help scientists to study the part of genome outside the genes-regulatory regions
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.
In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally .
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What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
Microarray -types, DNA chip, Principle and application of microarray, Preparation of DNA Chip, Affymetrix chip, microarray in genomics and proteomics, advantages and limitations of microarray
This presentation is fetures the basic introduction to Genome mosaicism in humans and nature, with some examples of its harmful effects on humans, with
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
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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.
2. DNA SEQUENCING
• Determining the precise order of nucleotides within a DNA molecule.
• Used to determine the sequence of individual genes, larger genetic
regions, full chromosomes or entire genomes.
• The resulting sequences may be used by researchers in molecular
biology or genetics to further scientific progress.
3. HISTORY OF DNA SEQUENCING
• 1972 – Earliest nucleotide sequencing – RNA sequencing of Bacteriophage
MS2 byWALTER FIESERR
• Early sequencing was performed with tRNA through a technique developed
by Richard Holley, who published the first structure of a tRNA in 1964.
• 1977 - DNA sequencing FREDRICK SANGER by Chain termination method
• Chemical degradation method by ALLAN MAXAM and WALTER GILBERT
• 1977 - First DNA genome t be sequenced of Bacteriophage ΦX174
• 1986 - LOREY and SMITH gave Semiautomated sequencing
• 1987 – Applied biosystems marketed Fully automated sequencing machines
4. •1995 – CRAIG VENTER, HAMILTON SMITH and collegues published first
complete genome sequence of Haemophilus influenzae
•2003 – Human genome project
•2ND Generation of DNA sequencing
•3RD Generation of DNA sequencing
5. Determining the Sequence of DNA
•Methods:
1) Maxam and Gilbert chemical degradation method
2) Chain termination or Dideoxy method
• Fredrick Sanger
3) Genome sequencing method
• Shotgun sequencing
• Clone contig approach
4) 2nd generation sequencing methods
• Pyrosequencing
• Nanopore sequencing
• Illumina sequencing
• Solid sequencing
6. SANGER SEQEUNCING
• Chain termination method of DNA sequencing.
• It involves following components:
a) 1. Primer
b) 2. DNA template
DNA polymerase
d) 4.. dNTPs(A,T,G,C)
e) 5. ddNTPs
• 4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis
8. Chain Termination (Sanger) Sequencing
ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
10. SANGER’S METHOD
• Not all polymerases can be used as they have mixed activity of
polymerizing and degrading.
• Both exonuclease activities are detrimental.
• Klenow fragment was used in orignal method but it has low
processivity.
• So Sequenase from bacteriophage T7 was uesd with high processivity
and no exonuclease added.
• Method requires ss DNA. So it is obtained by
• Denaturation with alkali or boiling
• DNA can be cloned in phagemid containg M13 ori and can take up DNA
fragments of 10kb
11. PYROSEQUENCING
• Pyrosequencing is the second important type of DNA sequencing methodology
in use today.
• The addition of a DNTP is accompanied by release of a molecule of pyrophosphate.
• Reaction mixture contains
DNA sample to be sequenced
Primers
Deoxynucleotides
DNA polymerase
Sulfurylase
• The release of pyrophosphate is converted by the enzyme sulfurylase into a flash
of chemiluminescence which is easily automated.
12. PYROSEQUENCING
Advantages:
Accurate
Parallel processing
Easily automated
Eliminates the need
for labeled primers
and nucleotides
No need for gel
electrophoresis
DISADVANTAGES
Smaller sequences
Nonlinear light
response after more
than 5-6 identical
nucleotides
13. MASSIVELY PARALLEL PYROSEQUENCING
• The DNA is broken down into fragments between 300 to 500bp
• Each fragment is ligated with a pair of adaptor
• To attach to the beads
• Provide annealing sites for the primers for performing PCR
• Adaptors are attached to beads by biotin-streptavidin linkage
• Just one fragment becomes attached to one bead
• Each DNA fragment is now amplified using
• PCR is carried out in a oil emulsion, each bead residing within own droplet
in the emulsion
• Each droplet contains all the reagents for PCR and is physically seprated
from all the other droplets by the barrier provided by the oil components
in the emulsion.
• After PCR, the droplets are transferred on wells on plastic strip and
pyrosequencing reactions are carried out
14.
15. SHOTGUN SEQUENCING
• Shotgun sequencing, also known as shotgun cloning, is a method used
for sequencing long DNA strands or the whole genome.
•In shotgun sequencing, DNA is broken up randomly into numerous small
segments and overlapping regions are identified between all the individual
sequences that are generated.
• Multiple overlapping reads for the target DNA are obtained by performing
several rounds of this fragmentation and sequencing.
•Computer programs then use the overlapping ends of different reads to
assemble them into a continuous sequence.
•The shotgun approach was first used successfully with the bacterium
Haemophilus influenzae.
•Craig venter used this method to map the Human genome project in 2001.
17. NEXT GENERATION SEQUENCING
• The concept behind NGS – the bases of small fragments of DNAare
sequentially identifed as signals emitted as eachfragment is
resynthesized from a dna template strand
• NGS extends this process across millions of reactions in a massively
parallel fashion rather than being limited to a single or a few dna
fragments
31. SOLiD SEQUENCING
• The SOLiD instrument utilizes a series of ligation and detection rounds to
sequence millions of fragments simultaneously.
• There are five primer cycles performed on the instrument with each
cycle staggered by a single base and including a series of seven or ten
ligations for either a 35 or 50 base pair sequencing run.
• Each ligation decodes two bases and is recorded through fluorescent
imaging.
• By compiling the fluorescent reads in color space for each fragment, an
accurate sequence can be generated.
• Two types of libraries are available for sequencing Fragment and Mate
Pairs.