The document discusses various genomic and proteomic tools and techniques that have revolutionized the field of microbial physiology. The advent of personal computers, the Internet, and rapid DNA sequencing techniques has fueled this renaissance by enabling widespread sharing of information among scientists. Genomic tools like gene cloning and sequencing provide insights into complete genetic instructions, while proteomic techniques examine dynamic protein expression and interactions. A variety of methods are described, including two-dimensional gel electrophoresis, mass spectrometry, and gene arrays.
Metagenomics is the study of genetic material recovered directly from environmental samples. Metagenomics is a molecular tool used to analyse DNA acquired from environmental samples, in order to study the community of microorganisms present, without the necessity of obtaining pure cultures.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Metagenomics is the study of genetic material recovered directly from environmental samples. Metagenomics is a molecular tool used to analyse DNA acquired from environmental samples, in order to study the community of microorganisms present, without the necessity of obtaining pure cultures.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Cell sorting is the process through which a particular cell type is separated from others
It is based on its differences in
Size, morphological parameters, viability protein expression
Applications including research, diagnosis, and therapy
cDNA library construction using mRNA which are derived from DNA. cDNA is formed from the reverse transcription of single stranded mRNA. cDNA contains only the exons, it donot not contains introns. The mRNA consists of poly A tail in which the tRNA and rRNA donot contains poly A tail. A short oligo nucleotide of Poly T is used to isolate mRNA seperately thereby single stranded mRNA is then converted into cDNA by using reverse transcriptase enzyme.
Genome annotation, NGS sequence data, decoding sequence information, The genome contains all the biological information required to build and maintain any given living organism.
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
Metagenomics is the study of metagenome, genetics material, recovered directly from environmental sample such as soil, water or faeces.
Metagenomics is based on the genomics analysis of microbial DNA directly
from the communities present in samples
Metagenomics technology – genomics on a large scale will probably lead to great advances in medicine, agriculture, energy production and bioremediation.
Metagenomics can unlock the massive uncultured microbial diversity present in the environment for new molecule for therapeutic and biotechnological application.
Metagenomic studies have identified many novel microbial genes coding for metabolic pathways such as energy acquisition, carbon and nitrogen metabolism in natural environments that were previously considered to lack such metabolism
Introduction to Synthetic Genome
SYNTHETIC GENOMICS Study of Invitro chemical synthesis of genetic material i.e., DNA in the form of oligonucleotides, genes, or genomes with Computational techniques for its design. SYNTHETIC GENOME Artificially synthesised genome (invitro)
Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. Metagenomics usually refers to the study of microbial communities.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
Cell sorting is the process through which a particular cell type is separated from others
It is based on its differences in
Size, morphological parameters, viability protein expression
Applications including research, diagnosis, and therapy
cDNA library construction using mRNA which are derived from DNA. cDNA is formed from the reverse transcription of single stranded mRNA. cDNA contains only the exons, it donot not contains introns. The mRNA consists of poly A tail in which the tRNA and rRNA donot contains poly A tail. A short oligo nucleotide of Poly T is used to isolate mRNA seperately thereby single stranded mRNA is then converted into cDNA by using reverse transcriptase enzyme.
Genome annotation, NGS sequence data, decoding sequence information, The genome contains all the biological information required to build and maintain any given living organism.
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
Metagenomics is the study of metagenome, genetics material, recovered directly from environmental sample such as soil, water or faeces.
Metagenomics is based on the genomics analysis of microbial DNA directly
from the communities present in samples
Metagenomics technology – genomics on a large scale will probably lead to great advances in medicine, agriculture, energy production and bioremediation.
Metagenomics can unlock the massive uncultured microbial diversity present in the environment for new molecule for therapeutic and biotechnological application.
Metagenomic studies have identified many novel microbial genes coding for metabolic pathways such as energy acquisition, carbon and nitrogen metabolism in natural environments that were previously considered to lack such metabolism
Introduction to Synthetic Genome
SYNTHETIC GENOMICS Study of Invitro chemical synthesis of genetic material i.e., DNA in the form of oligonucleotides, genes, or genomes with Computational techniques for its design. SYNTHETIC GENOME Artificially synthesised genome (invitro)
Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. Metagenomics usually refers to the study of microbial communities.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene.
Class9 DNA technology in secondary schoolssusera700ad
Biotechnology is the use of an organism, or a component of an organism or other biological system, to make a product or process.
Many forms of modern biotechnology rely on DNA technology.
DNA technology is the sequencing, analysis, and cutting-and-pasting of DNA.
Common forms of DNA technology include DNA sequencing, polymerase chain reaction, DNA cloning, and gel electrophoresis.
Biotechnology inventions can raise new practical concerns and ethical questions that must be addressed with informed input from all of society.
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
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
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
Wastewater treatment is a process used to remove contaminants from wastewater and convert it into an effluent that can be returned to the water cycle. Once returned to the water cycle, the effluent creates an acceptable impact on the environment or is reused for various purposes (called water reclamation).
The treatment process takes place in a wastewater treatment plant. There are several kinds of wastewater which are treated at the appropriate type of wastewater treatment plant. For domestic wastewater (also called municipal wastewater or sewage), the treatment plant is called a sewage treatment plant. For industrial wastewater, treatment either takes place in a separate industrial wastewater treatment plant, or in a sewage treatment plant (usually after some form of pre-treatment). Further types of wastewater treatment plants include agricultural wastewater treatment plants and leachate treatment plants.
To minimize the initial generation of waste materials through source reduction, then through reusing and recycling to further reduce the volume of the material being sent to landfills or incineration compared to the conventional approach of simply focusing on disposal of solid waste.
Biowaste is all types of waste that contains or is likely to contain hazardous biological agents (= live infectious or otherwise hazardous micro-organisms).
Biowaste is mainly generated in diagnostic and research laboratories ("biosafety labs"), as well as in hospitals (biowaste thus includes part of the hospital waste).
Patients suffering from COVID-19 displayed hypocalcemia, vitamin D deficiency, and are often immobilized due to the disease, which will contribute to bone demineralization.
Moreover, the patients regularly present with joint and muscle pain. These symptoms resemble early aging characteristics observed during the development of OA.
Although no viral presence in the joint could be observed, early aging environmental changes could contribute to the development of this early OA-like phenotype.
Drugs that target the renin-angiotensin and immune system were explored as a treatment option for COVID-related musculoskeletal alterations.
This includes RAS modulating therapy, circadian clock modulators, immunomodulators, and chronotherapy
We believe that SARS-CoV-2 contributes to early aging perturbations, such as endothelial and adipose tissue dysfunction, the most frequent long-lasting symptoms of SARS-CoV-2 infection as the result of this accelerated aging process.
Lung cancer is the most common cancer in males and second most common in females after breast cancer.
it is the third most commonly diagnosed and leading cause of cancer death in Pakistan, with an estimated 6,800 (4.6%) new cases and 6,013 (5.9%) deaths occurring in 2012
We have compared our data with the international statistics to see where do we stand.
In Pakistan, we do not have a valid central cancer registry at present which can provide a true picture of lung cancer. This calls for an urgent need to formulate a valid central cancer registry in the country in association with the local bodies.
Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus.
Most people who fall sick with COVID-19 will experience mild to moderate symptoms and recover without special treatment.
HOW IT SPREADS
The virus that causes COVID-19 is mainly transmitted through droplets generated when an infected person coughs, sneezes, or exhales. These droplets are too heavy to hang in the air, and quickly fall on floors or surfaces.
You can be infected by breathing in the virus if you are within close proximity of someone who has COVID-19, or by touching a contaminated surface and then your eyes, nose or mouth.
Microbial fuel cells are devices that use bacteria to turn the energy stored in chemical bonds into electrical current that we can use without the need for combustion. Essentially, we are harnessing the power of metabolism for electricity.
“Cytogenetics traditionally refers to the study of chromosomes by microscopy following the application of banding techniques, permitting identification of abnormalities of chromosome number, loss or gain of chromosomal material or positional changes”. Standardized nomenclature is critical for the accurate and consistent description of genomic changes as identified by karyotyping, fluorescence in situ hybridization and microarray. The International System for Human Cytogenomic Nomenclature (ISCN) is the central reference for the description of karyotyping, FISH, and microarray results, and provides rules for describing cytogenetic and molecular cytogenetic findings in laboratory reports. These laboratory reports are documents to the referring clinician, and should be clear, accurate and contain all information relevant for good interpretation of the cytogenetic findings.
This presentation is all about fuels & industrial chemicals.
Important highlights of this presentations are following
*Importance Of Organic Compounds in Fuel Industry
» Introduction of Organic Compounds & Organic Acids
» Why Organic Acids are of interest??
» Role of Organic Chemistry in oil industry
» Organic compound of gasoline
» Alkanes & Importance Of Alkanes
» Bioconversion of Maize Starch
» Starch
» Bioconversion
» Bio products From Starch Waste
» Maize & it’s Products
» Important Products From Maize
» Process OF Bioconversion Of Maize
» Fungal biomass protein production from Starch processing Water
» Ethanol production from various substrates
» Ethanol production from barley β-glucan by yeast displaying Aspergillus
» Ethanol production in solid substrate fermentation using thermo tolerant yeast
» Ethanol production by solid state fermentation of sweet sorghum using thermo tolerant yeast strain
» Ethanol production from agricultural biomass substrates
» Ethanol production From Starch & Molasses
» Industrially Important Amino Acids Production
» l-Lysine production & its uses
» Citric Acid Production & its uses
» Guluconic Acid Production & its uses
» Lactic Acid production & its Uses.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
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.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
3. Contents
• Genomic & proteomic tools
• Cloning a Genome
• DNA sequencing
• Internet Tools for DNA
sequencing
• Gene Replacement
• Gene Arrays
• Proteomics
• Traditional tools
• Reporter genes
• DNA mobility Shifts
• Primer Extension
• Detecting DNA ,RNA proteins by
Southern & western Blots
• Two-Hybrid Analysis
4. Introduction
• The field of microbial physiology is undergoing a
dramatic revolution. This reformation is the direct
result of three technological achievements:
• The personal computer,
• The Internet
• rapid DNA sequencing techniques
5. Advantages
• The personal computer and Internet have fueled this
renaissance of microbial physiology by allowing
unfettered sharing of this information among scientists
around the world.
• Rapid DNA techniques, are useful in synthetic biology as in
metabolic engineering in plants & Microbes
• They are also useful in forensic techniques, as increase
performance of human identification processes,
6. Genomics &
Proteomics
• Genomics provides an overview of
the complete set of genetic
instructions provided by the DNA,
while transcriptomics looks into gene
expression patterns.
• Proteomics studies dynamic protein
products and their interactions, while
metabolomics is also an intermediate
step in understanding organism's
entire metabolism.
7. Why Genomics is important??
• Genetics involves scientific studies of genes and their effects.
... Genomics includes the scientific study of complex diseases
such as heart disease, asthma, diabetes, and cancer because
these diseases are typically caused more by a combination of
genetic and environmental factors than by individual genes
8. Use of proteomics
• Proteomics is used to detect protein expression
patterns at a given time in response to a specific
stimulus, but also to determine functional protein
networks that exist at the level of the cell, tissue, or
whole organism
12. Gene Cloning: The insertion of a fragment of DNA carrying a
gene into a cloning vector and subsequent propagation of
recombinant DNA molecules into many copies is known as
gene cloning.
14. BASIC STEPS OF GENE CLONING
Construction of recombinant DNA molecule
Transport of the recombinant DNA to the host cell
Multiplication of recombinant DNA molecule
Division of the host cell
Numerous cell division resulting in a clone
15. Gene cloning requires specialized tools and
techniques:
Vehicles: The central component of a gene cloning
experiment is the vehicle, which transport the gene into the
host cell and is responsible for its replication. To act as a
cloning vehicle a DNA molecule must be capable of entering
a host cell and, once inside, replicating to produce multiple
copies of itself.
Vector: A DNA molecule, capable of replication in a host
organism, into which a gene is inserted to construct a
recombinant DNA molecule.
16. Different types of cloning
1. Recombinant DNA technology or DNA
cloning
2. Reproductive cloning and
3. Therapeutic cloning
17. How can cloning technologies be used?
• Gene therapy can be used to treat certain genetic conditions by
introducing virus vectors that carry corrected copies of faulty genes
into the cells of a host organism.
• Genes from different organisms that improve taste and nutritional
value or provide resistance to particular types of disease can be used
to genetically engineered food crops.
•Reproductive cloning also could be used to repopulate endangered
animals or animals that are difficult to breed.
18. Can organs be cloned for use in transplants?
Scientists hope that one day therapeutic cloning can be used to
generate tissues and organs for transplants.
The stem cells would be used to generate an organ or tissue that
is a genetic match to the recipient.
In theory, the cloned organ could then be transplanted into the
patient without the risk of tissue rejection.
20. Objectives
• Compare and contrast the chemical (Maxim/Gilbert) and chain
termination (Sanger) sequencing methods.
• List the components and molecular reactions that occur in chain
termination sequencing.
• Discuss the advantages of dye primer and dye terminator
sequencing.
• Derive a text DNA sequence from raw sequencing data.
• Describe examples of alternative sequencing methods, such as
bisulfite sequencing and pyrosequencing.
22. Maxam-Gilbert sequencing is performed by chain breakage
at specific nucleotides.
DMS
G
G
G
G
FA
G
A
G
G
A
G
A
A
H
C
T
T
C
T
C
C
T
H+S
C
C
C
C
Maxam-Gilbert Sequencing
23. Sequencing gels are read from bottom to top (5′ to 3′).
G G+A T+C C
3′
A
A
G
C
A
A
C
G
T
G
C
A
G
5′
Longer fragments
Shortest fragments
G
A
Maxam-Gilbert Sequencing
24. Chain Termination (Sanger) Sequencing
• A modified DNA replication
reaction.
• Growing chains are
terminated by
dideoxynucleotides.
25. Chain terminates
at ddG
Chain Termination (Sanger) Sequencing
The 3′-OH group necessary for formation of the phosphodiester bond is
missing in ddNTPs.
26. Template area to be sequenced
-3′ OH
TCGACGGGC…
5′OP-
Primer
Template
Chain Termination (Sanger) Sequencing
• A sequencing reaction mix includes labeled primer and
template.
• Dideoxynucleotides are added separately to each of the
four tubes.
27. 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
Chain Termination (Sanger) Sequencing
28. Chain Termination (Sanger) Sequencing
• With addition of enzyme (DNA polymerase), the primer
is extended until a ddNTP is encountered.
• The chain will end with the incorporation of the ddNTP.
• With the proper dNTP:ddNTP ratio, the chain will
terminate throughout the length of the template.
• All terminated chains will end in the ddNTP added to
that reaction.
29. Chain Termination (Sanger) Sequencing
• The collection of fragments is a sequencing ladder.
• The resulting terminated chains are resolved by
electrophoresis.
• Fragments from each of the four tubes are placed in
four separate gel lanes.
30. Sequencing gels are read from bottom to top (5′ to 3′).
G A T C
3′
G
G
T
A
A
A
T
C
A
T
G
5′
Longer fragments
Shorter fragments
ddG
ddG
Chain Termination (Sanger) Sequencing
31. Cycle Sequencing
• Cycle sequencing is chain termination sequencing
performed in a thermal cycler.
• Cycle sequencing requires a heat-stable DNA
polymerase.
32. Alternative Sequencing Methods:
Pyrosequencing
• Pyrosequencing is based on the generation of light
signal through release of pyrophosphate (PPi) on
nucleotide addition.
• DNAn + dNTP DNAn+1 + PPI
• PPi is used to generate ATP from adenosine
phosphosulfate (APS).
• APS + PPI ATP
• ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.
33. DNA sequence: A T C A GG CC T
Nucleotide added : A T C A G C T
Alternative Sequencing Methods:
Pyrosequencing
• Each nucleotide is added in turn.
• Only one of four will generate a light signal.
• The remaining nucleotides are removed enzymatically.
• The light signal is recorded on a pyrogram.
34. Alternative Sequencing Methods:
Bisulfite Sequencing
• Bisulfite sequencing is used to detect
methylation in DNA.
• Bisulfite deaminates cytosine, making uracil.
• Methylated cytosine is not changed by
bisulfite treatment.
• The bisulfite-treated template is then
sequenced.
35. Annotation
• DNA annotation or genome annotation is the
process of identifying the locations of genes and
all of the coding regions in a genome and
determining what those genes do. ... Once
a genome is sequenced, it needs to
be annotated to make sense of it
37. How to handle the large amount of
information?
• Answer: bioinformatics and Internet
Drew Sheneman, New Jersey--The Newark Star Ledger
38. Introduction: What is bioinformatics?
• Bioinformatics is a scientific discipline created from the interaction of
biology and computer science.
• Can be defined as the body of tools, algorithms needed to
handle large and complex biological information.
• The NCBI defines bioinformatics as:
"Bioinformatics is the field of science in which biology, computer
science, and information technology merge into a single discipline”
39. • There is a need for computers and algorithms that
allow:
Access, processing, storing, sharing,
retrieving, visualizing, annotating…
• But!!!!
•Why do we need the Internet?
40. • “omics” projects and the information associated
with involve a huge amount of data that is stored
on computers all over the world.
• Because it is impossible to maintain up-to-date
copies of all relevant databases within the lab.
Access to the data is via the internet.
41.
42.
43.
44. DNA (nucleotide sequences) databases
• They are big databases and searching either one should produce similar
results because they exchange information routinely.
• -GenBank (NCBI): http://www.ncbi.nlm.nih.gov
• -Ensembl: http://useast.ensembl.org/index.html
•
• -DDBJ (DNA Database of Japan): http://www.ddbj.nig.ac.jp
• -TIGR: http://tigr.org/tdb/tgi
• -Yeast: http://yeastgenome.org
• -Microbes: http://img.jgi.doe.gov/cgi-bin/pub/main.cgi
45. Protein (amino acid) databases
• Known proteins:
-Swiss-Prot (very high level of annotation)
http://au.expasy.org/
-PIR (protein identification resource) the world's most
comprehensive catalog of information on proteins http://www.pir.uniprot.org/
• Translated databases:
-TREMBL (translated EMBL): includes entries that have not been
annotated yet into
Swiss-Prot. http://www.ebi.ac.uk/trembl/access.html
-GenPept (translation of coding regions in GenBank)
-pdb (sequences derived from the 3D structure
• Brookhaven PDB) http://www.rcsb.org/pdb/
46. Which algorithm to use for database similarity
search?
• Speed:
• BLAST > FASTA > Smith-Waterman (It is very slow and uses a lot of
computer power)
• Sensitivity/statistics:
• FASTA is more sensitive, misses less homologues
• Smith-Waterman is even more sensitive.
• BLAST calculates probabilities
• FASTA more accurate for DNA-DNA search then BLAST
50. Reverse Genetics
• From gene to phenotype - using genetic tools to
identify the function of a gene without prior
knowledge of its function.
• Knockout - screen for phenotype
• Overexpression
• Ectopic expression
54. Gene Array
• A snapshot that captures the activity pattern of
thousands of genes at once
55. Basics of microarrays
• DNA attached to solid
support
Glass, plastic, or nylon
• RNA is labeled
Usually indirectly
• Bound DNA is the probe
Labeled RNA is the “target”
60. Definition:
That’s just not a protein biochemistry !
Proteome :
It is the complement protein found in a single cell in a
particular environment./ is complete collection of proteins encoded by
genome of an organism.
Proteomics :
It is the study of composition, structure, function and
interaction of the proteins directing the activities of each living cell.
61. Role of Proteomics
• To study the structure and function of protein.
• To study the 3D structure of protein.
• Study of qualitative and quantitative analysis of proteins.
67. Introduction:
•Two-dimensional gel electrophoresis
is considered a powerful tool for
proteomics work.
• It is used for separation and
fractionation of complex protein
mixtures from biological samples.
Two dimensional Gel Electrophoresis
68. The first one is called isoelectric
focusing (IEF) which separates
proteins according to
isoelectric points (pI)
The second step is SDS-
polyacrylamide gel
electrophoresis (SDSPAGE) which
separates proteins based on the
molecular weights
•Thus, thousands of proteins
can be separated, and the
information about IEF and
molecular weights can be
obtained
Steps
69. •Proteins are amphoteric
molecules and the positive,
negative, or zero net charge they
carry depending on the pH of the
surroundings.
•The isoelectric point (pI) is
defined as the pH of a solution at
which the net charge of the
protein becomes zero.
Isoelectric Focusing (IEF)
70. • A protein with a positive net charge will migrate toward the
cathode, becoming less positively charged until reaching its
pI. While a protein with a negative net charge will migrate
toward the anode, becoming less negatively charged until it
also reaches its pI.
71. Isoelectric Focusing (IEF)
• A protein mixture is loaded at
the basic end of the pH
gradient gel.
• After applying an electric
field, the proteins are
separated depending on
charges, focusing at positions
where the pl value is
equivalent to the surrounding
pH.
• Larger proteins will move
more slowly through the gel,
but with sufficient time will
catch up with small proteins of
equal charge
72. • Be performed on flatbed or vertical systems on a slab
gel.
• The second dimension is often performed by SDS-
PAGE), which is an electrophoretic method.
• This technique is used to separate proteins by their
molecular weight.
• SDS negatively charged detergent used to denature
proteins.
SDS-PAGE
(SDS-polyacrylamide gel electrophoresis)
73. How does an SDS-PAGE Seperation Work?
• Negatively charged proteins move to positive Electrode.
• SDS coated larger proteins migrate slowly through the Gel
Matrix.
• SDS coated smaller proteins
migrate quickly through the Gel Matrix.
76. Coomassie Blue staining
• A relatively simple method and more quantitative than silver
staining.
• It is suitable to detect protein bands containing about 0.2μg
or more proteins.
77. MASS SPECTROMETRY
• While 2D- gel electrophoresis separates proteins, it doesn’t
identify them.
• MS is used to identify them which separates charged
particles or ions according to mass.
78. 3 Major Parts
• Source ionized the sample.
• Analyzer separate the ions on m/z ratio.
• Detector sees the ions and analyzed the result.
79. How does a Mass Spectrometer
work?
• Samples easier to manipulate if ionised.
• Separation in analyser according to mass-to-charge ratios
(m/z).
• Detection of separated ions and their relative abundance.
• Signals sent to data system and formatted in a m/z spectrum .
80.
81. •The study of proteins is very complex because the
concentration of protein is different in each organism
and in each cell of the organism.
Complexities in proteomics
82.
83. • Shows that genetic alterations are not the
reason for all types of diseases.
• Helps in determining the proper treatment of
diseases.
• With the help of three dimensional analysis of
proteins we have found that HIV protease is the
enzyme which is responsible for AIDS.
Advantages of study of proteomics
84. Advanced screening for disease.
• One of the most important use of
proteomics in diagnosis is the
identification of biomarkers.
• The study of drugs in proteomics is
called pharmacoproteomics
85. Biomarkers
• Biomarkers are molecules that indicate normal or
abnormal process taking place in your body and may
be a sign of an underlying condition or disease.
• DNA (genes), proteins or hormones, can serve as
biomarkers, since they all indicate something about
your health.
86. TRADITIONAL TOOLS
• Mutant Hunts:
Used to expose the details of a biochemical pathway
requires the presence of a selectable phenotype.
Mutants that have lost this phenotype are then sought.
Once a mutant is found:
1. Encoding gene can be mapped.
2. Identified.
3. Cloned.
4. Sequenced.
87. Positive Selection
• Only mutants that fail to transport or use
the amino acid analog will grow and form
colonies on a plate containing that analog.
• In looking for mutants defective in
carbohydrate fermentation, mutant colonies
that fail to ferment the test sugar will
appear white among many no mutated red
colonies.
88. Negative selection
• The phenotype of an E. coli
mutant that cannot make the
amino acid alanine will only grow
on a defined medium if that
medium contains alanine.
90. • Very powerful tool for analyzing various
aspects of gene expression involves
fusing easily assayed reporter genes such
as lacZ (ß-galactosidase) or gfp (green
fluorescent protein) to host target gene
promoters.
91. Reporter genes
• Reporter gene is defined as a gene
whose products detects or marks the
cells, tissues, organisms that express
the gene from those that do not.
92.
93. • Reporter genes isolated from prokaryotes, E.coli,
are used in fishes-lacZ gene,
Cat(Chloramphenicol Acetyl Transferase gene).
• Recently luciferase and green fluorescent protein
are used.
94. Functions
• Encode for the products that can
be easily quantified in
transformed tissues by simple
assays.
95.
96. • Also help to detect transformation
events during gene transfer
experiment.
• Encode for the products that are
themselves fluorescent or give
fluorescence reactions upon
treatment with other substrates.
97. Types of reporter genes
Scorable reporter genes:
Expression of this result in
quantified phenotype.
Easily detected by highly
sensitive enzyme assays.
Selectable reporter
genes:
Expression of resistance to
Toxin.
Selection of transformants
in growth media containing
selective agent.
98. How does lacZ REPORTER GENE
WORK?
• lacZ gene encodes the beta-galactosidase,
which catalyzes the cleavage of lactose to
form galactose and glucose.
• Reporter gene can be used under the
control of promoter or enhancer.
99. Types of Fusion
• There are two types of fusion
• 1st type;
Operon fusion
• 2nd type;
Gene fusion
100.
101. Operon fusion
• Operon or transcriptional fusion are used when a
promoter less lacZ gene are inserted within a
target gene.
• This occurs in an orientation that places lacZ under
the control of target gene promoter.
102. • Factors control expression from the
target gene also control the
production of ß-galactosidase..
• Since lacZ message resulting from
fusion still contains its ribosome
binding site.
• Observe regulation is typical, but not
always due to transcriptional control.
103. Gene fusion
• 2nd type known as gene or protein fusion.
• Involves inserting a reporter gene lacZ into
target gene that is missing its both ribosome
binding site and promoter.
104. • Messages from the target and reporter gene fused.
• Truncated target gene peptide and reporter gene peptide
also fused when inserted in a proper reading frame
In this case, anything controlling the transcription and
translation of target gene will also control the ß-galactosidase
levels.
105. • Construction of these fusions sometimes
involves using a genetically engineered µ phage
to randomly insert reporter lacZ gene into E.coli
or Salmonella genome.
106.
107. • Alternatively, the fusion can be constructed in vitro and
transferred into recipient cell.
• Once constructed these fusions, allow for monitoring of
how different growth conditions or known regulators
influence transcription/translation of the gene.
108. DNA mobility shifts
• Once a gene encoding a putative transcriptional regulatory
protein is identified whether the protein directly binds to
target DNA sequence.
• One approach is to take purified protein
• Add it to putative target DNA fragment
• See if the protein cause of shift in the electrophoretic
mobility of fragment.
109.
110. • Linear DNA undergoing electrophoresis in an agarose
or polysaccharide gel matrix will travel at the rate
based on size of DNA fragment.
• Larger molecules travel slower than the smaller
molecules.
111. • If protein is bind to DNA fragment complex
will be large and travel slowly.
• This is called mobility shift or GEL shift.
112. EMSA
• Electrophoretic mobility shift assay
• Method used for analyzing protein DNA interaction.
• Simple, efficient and sensitive technique
• DNA moves through the gel faster when not bound to protein.
• Used to identify DNA binding proteins.
113.
114. Super shift
• A super shift of the target fragment will occur
when an antibody directed against putative DNA
binding protein into binding reaction.
• Complex of anti DNA binding protein , DNA binding
protein and DNA is even larger than binding protein
DNA complex causing greater gel shift.
117. HISTORY
• As is the photo copier a basic requirement in
an office ,so is the PCR machine in a
molecular biology Laboratory !!!!!!!!!
• PCR is DNA replication in a test
Tube…….
Great mind behind this PCR :Kary Banks
Mullis
Developed PCR in 1985 and was awarded
Nobel prize in 1993.
PCR machine otherwise called Thermo
cycler.
119. Polymerase Chain Reaction(PCR)
• PCR targets and
amplifies a specific
region of a DNA
strand.
• It is an in vitro
technique to generate
large quantities of a
specified DNA
120. SAMPLE OF DNA
• Often, only a small amount of DNA is available e.g. drop of blood, Semen strains,
• Single hair, vaginal swabs etc.
• Two methods currently exist for amplifying the DNA or making Copies.
• Cloning—takes a long time for
enough clones to reach maturity.
• PCR—works on even a
single molecule quickly
124. Factors for Optimal PCR
1.PCR Primers
-correctly designed pair of primers is
required
-primer dimer , hairpin formation should be
prevented
-length of primer
125. 2.DNA Polymerase
-Thermus aquaticus-170° F
-Taq polymerase is heat resistant
-It lacks proof reading exonuclease activity
-Other polymerases can be used .eg:
Tma DNA Polymerase from Thermotoga maritama,
Pfu DNA Polymerase from Pyrococcus furiosus
126. 3.Annealing Temperature
• Very important since the success and specificity of PCR depend on
it because DNA-DNA hybridization is a temperature dependent
process.
• If annealing temperature is too high ,pairing between primer and
template DNA will not take place then PCR will fail.
• Ideal Annealing temperature must be low enough to enable
hybridization between primer and template but high enough to
prevent amplification of no target sites.
• Should be usually 1-2° C or 5° C lower than melting temperature of
the template-primer duplex
127. 4.Melting Temperature
• Temperature at which 2 strands of the duplex dissociate.
• It can be determined experimentally or calculated from
formula
• Tm = (4(G+C)) + (2(A+T))
G/C content
- ideally a primer should have a near random mix of
nucleotides, a 50% GC content
- there should be no PolyG or PolyC stretches that can
promote non-specific annealing
128. TYPES OF PCR
• Anchored PCR
• Reverse transcriptase PCR
• RACE PCR
• Quantitative real time PCR (Q-RT PCR)
• Asymmetric PCR
• Allele- Specific PCR
129. APPLICATION OF PCR
1.Molecular Identification
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
133. Primer extension.
1
2
3
• It is a method that is used
to map 5’ end of an RNA
• It determines the start site of transcription
• It provides the evidence
where the promoter is
located within a cloned
gene.
134. Primer extension .
RNA from genes have multiple
promtor that produce different-
sized primer extension
fragments.
The size of the primer extension
fragmets will be equal to one of
the “rungs”on the ladder.
Radioactive nucleotides are used
to radiolabelled the cDNA.
Reverse transcriptase+dNTPs
are added that extend the primer
at 5’ end of RNA
A DNA primer annels to the
mRNA that is complemtary to it
at 5’end.
The primer extension fragments
are separated by gel
electrophoreisis on
polyacrylamide gel.
137. Southern Blotting.
• A British biologist Edwin Southern developed this technique in 1975.
Definition.
“It is a technique that is used to detect DNA in a sample ,
and determine how much DNA is present”.
138. Mechanism.
.GEL ELECTROPHORESIS.
The fragments are separated according
to their molecular weight on agarose gel
by electrophoresis.
.BLOTTING.
The fragments of DNA are
transferred to a sheet of nylon
or nitrocellulose, which hold
the fragments immobile.
.WASHING.
.The unhybridized or unbound
probe is removed by washing with
buffer.
.PROBE LABELING.
.DNA/RNA EXTRACTION.
DNA which has been extracted from
cells is purified.
.AUTORADIOGRAPH.
After anneling the DNA fragments are visualized
by exposing the membrane to X-ray film.
.RESTRICTION ENZYME.
DNA is cleaved into fragments
by the enzyme knwn as
Restriction endonucleases.
.HYBRIDIZATION.
The labelled probe is incubated with the
complementary DNA sequence to form a
section of double stranded DNA, or to
promote hybridization of complemetary
sequences.
1
5
2
6
8
4
7 3
A nucleic acid probe with sequence homologous to the target
sequence is labelled with flourescent dye, biotin or enzyme
that generate chemiluminescent signal. .
141. Northern Blotting.
Definition.
“It is a technique that is used to identify RNA in a sample
on the basis of their size ,abundance, and processing”.
• This technique was developed in 1977 by James Alwine ,
David Kemp , and George Stark.
142. Mechanism.
NORTHERN
BLOTTING.
5
6
4
1
3
2
Washing.
Phosphorimaging.
The fragments are visualized
or autoradiographed
Probe labelling.
Radiolabeled DNA probe is
used to probed the membrane.
Gel electrophoresis.
The RNA is loaded on agarose
formaldehyde gel for
fractionation.
RNA isolation.
RNA is extracted from the cell
with RNases
Blotting.
The fragments are trasferred to nylon
or nitrocellulose membrane.
.The unhybridized or unbound
probe is removed by washing with
buffer.
145. Western Blotting.
Definition.
“A technique that is used for detecting specific proteins
separated by electrophoresis by the use of antibodies in
a given sample of tissue or extract”.
This technique was also developed by Edwin Southern in 1977.
146. Mechanism.
WESTERN
BLOTTING.
5
6
4
1
3
2
Washing.
The unbound or unhybridized
probe is removed by washing
Autoradiography.
. The addition of hydrogen
peroxide and luminol creates a
luminescent light
Probe labelling.
Antibodies “sandwich” is used
to probe the membrane.
Gel electrophoresis.
The protein is loaded on
agarose gel for fractionation.
Protein extraction.
Proteins are extracted from the
tissue of the given cell
culture(MOUSE,RABBIT)
BLOTTING.
The fragments are transferred to
nylon or nitrocellulose
06
01
02
03
04
05
149. Southwestern Blot.
Definition.
“It is a technique that identifies DNA binding proteins by
their ability to bind to specific oligonucleotide probe”.
It was first described by B. Bowen, Steinberg and
colleagues in 1980.
150. Mechanism.
Southwestern blot.
Protein bands bind to labeled
DNA is visualized by
Autoradiography.
The unbound or unhybridized
probe is remove by washing.
Radiolabeled Oligonucleotides
are used to probe the membrane
.
Proteins are separated using
standard SDS polyacrylamide gel
electrophoresis.
Protein that binds specific
DNA molecule are
extracted.
The fractionated protein bands
are transferred to a nylon or
nitrocellulose membrane.
152. Two-Hybrid Analysis
• Protein–protein interactions are an important part of many
regulatory circuits.
• Two-hybrid systems are cleverly designed tools that not
only measure in vivo protein–protein interaction
• Can be used to mine for unknown “prey” proteins that
interact with a known “bait” protein.
153. Using the Two-Hybrid System to Identify a
New Regulator
• Helicobacter pylori, a remarkable gram-
negative microbe that can inhabit the
human stomach and cause gastric
ulcers
• Provides an interesting example in
which a two-hybrid approach was used
in conjunction with proteomic and
genomic strategies to reveal a new
family of regulators
154. Common Methods to Analyze Protein-
Protein Interactions
• Biochemical and biophysical
approaches
• Affinity chromatography
• Co immunoprecipitation
• Fluorescence Resonance
Energy Transfer (FRET)
• Surface Plasma Resonance
• Atomic Force Microscopy
(AFM)
• X-ray Diffraction Molecular
genetic approaches
• Yeast Two-hybrid
155. Overall summary of Yeast two-hybrid
experiment
• Yeast two-hybrid experiments yield information on
protein protein interactions
• GAL4 Binding Domain
• GAL4 Activation Domain
• X and Y are two proteins of interest
•If X & Y interact then reporter gene is expressed
156.
157. Major applications of classical
system
• Used to determine whether two known proteins
interact with one another.
• Used to identify unknown proteins, encoded by a
cDNA library, that interact with a protein of
interest.
• Powerful tool for investigating the network of
interactions that form between proteins involved in
particular biological processes
158. Reporter Genes
• LacZ reporter - Blue/White Screening
• HIS3 reporter - Screen on His+ media (usually need
to add 3AT to increase selectivity)
• LEU2 reporter - Screen on Leu+ media
• ADE2 reporter - Screen on Ade+ media
• URA3 reporter - Screen on Ura+ media (can do
negative selection by adding FOA
159. Modifications of the Yeast Two-
Hybrid system
• The hSos recruitment system
• Three protein system
• The dual-bait system
• The reverse two-hybrid system
160. Uses of the Yeast Two-Hybrid System
• Identification of caspase substrates
• Interaction of Calmodulin and L-Isoaspartyl
Methyltransferase
• Genetic characterization of mutations in E2F1
• Peptide hormone-receptor interactions
• Pha-4 interactions in C. elegans
161. Summary
• There are numerous commonly used molecular tools for
studying microbial physiology.
• With the help of above genomic & proteomic tools molecular
biologists use to examine ,DNA sequences, gene expression,
DNA–protein interactions, and protein–protein interactions
• Or when sequence of a complete genome is obtained and the
means by which that knowledge can be used to study microbial
physiology and evolution