A gene mutation (myoo-TAY-shun) is a change in one or more genes. Some mutations can lead to genetic disorders or illnesses. A gene can mutate because of a change in one or more nucleotides of DNA, a change in many genes, loss of one or more genes, rearrangement of genes or whole chromosomes.
It highlights the various methods of gene transfer in plants, characterization of plants by PCR and qRTPCR. Different types of PCR and Real time PCR have been described
Protein engineering is the process of developing useful or valuable proteins.
Protein Engineering is a second generation of recombinant DNA technology.
It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties.
Site directed mutgenesis, OLIGONUCLEOTIDE DIRECTED MUTAGENESIS Vipin Shukla
INTRODUCTION, HISTORY, MUTATION, DIRECTED MUTAGENESIS,BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS,METHOD FOR SITE DIRECTED MUTATIONS,THE SINGLE PRIMER METHOD, CASETTEE MUTAGENESIS, PCR-SITED DIRECTED MUTAGENESIS, APPLICATION OF SITE DIRECTED MUTAGENESIS.
It highlights the various methods of gene transfer in plants, characterization of plants by PCR and qRTPCR. Different types of PCR and Real time PCR have been described
Protein engineering is the process of developing useful or valuable proteins.
Protein Engineering is a second generation of recombinant DNA technology.
It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties.
Site directed mutgenesis, OLIGONUCLEOTIDE DIRECTED MUTAGENESIS Vipin Shukla
INTRODUCTION, HISTORY, MUTATION, DIRECTED MUTAGENESIS,BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS,METHOD FOR SITE DIRECTED MUTATIONS,THE SINGLE PRIMER METHOD, CASETTEE MUTAGENESIS, PCR-SITED DIRECTED MUTAGENESIS, APPLICATION OF SITE DIRECTED MUTAGENESIS.
Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010TigerTox
Historical and current perspectives on genetic toxicology, with commentary and slides on assay predictivity and shortcomings, regulatory guidance, and high-throughput screens to enhance preclinical drug safety.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
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.
Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010TigerTox
Historical and current perspectives on genetic toxicology, with commentary and slides on assay predictivity and shortcomings, regulatory guidance, and high-throughput screens to enhance preclinical drug safety.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
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.
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.
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.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
2. Content
Gene Mutation
Site-Directed Mutagenesis
Mutagenic Agents and Consequences of Mutation
Epigenetics
Genetic Recombination
Genetic Diseases and Gene Therapy
Cell Cycle (Phases and Biochemical Checkpoints)
3. Gene Mutation
Side-directed Mutagenesis
Mutagenic Agents and Consequences of Mutation
Epigenetics
Genetic Recombination
Genetic Diseases and Gene Therapy
Cell Cycle (phases and biochemical checkpoints)
4. Mutation
Alteration of the nucleotide sequence of the
genome of an organism, virus, or
extrachromosomal DNA.
Can be transmitted to descendants
May or may not produce discernible changes
in the phenotype (observable characteristics)
of an organism
may be spontaneous (natural) due to
mistakes in DNA replication, recombination,
and nuclear division, or induced (artificial)
5. Classification based on structural changes
1. Genomic mutation: This involves changes in
chromosome number (gain or loss in complete
sets of chromosomes or parts of a set)
2. Structural mutation: This has to do with
changes in chromosome structure e.g.
duplication of segments, translocation of
segments
3. Gene mutation: This refers to changes in the
nucleotide constitution of DNA by deletion or
substitution
6.
7. Mutations that convert the wild-type (the common phenotype) to the mutant
form (the rare phenotype) are called forward mutations, while those that
change a mutant phenotype to a wild-type are called reverse mutations
Wild-type Mutant
8. Molecular Nature of Mutation
Point Mutations (Base substitution):
Point mutations are those mutations due to the substitution of one
base pair for another. They may be either transitions or transversions.
Transition: This is a form of point mutation where a
purine base replaces another purine base or a
pyrimidine base replaces another pyrimidine base
within a DNA
Transversion: This involves the replacement of a purine
by a pyrimidine, or vice versa.
12. Gene Mutation
Side-directed Mutagenesis
Mutagenic Agents and Consequences of Mutation
Epigenetics
Genetic Recombination
Genetic Diseases and Gene Therapy
Cell Cycle (phases and biochemical checkpoints)
13. Site-directed Mutagenesis (SDM)
Site-directed mutagenesis is a molecular
biology method that is used to make specific
and intentional changes to the DNA sequence
of a given gene and any gene products.
It is one of the most important techniques in
laboratory for introducing a mutation into a
DNA sequence.
It is used for investigating the structure and
biological activity of DNA, RNA, and protein
molecules, and for protein engineering.
14. Methods for Site-directed Mutagenesis
SDM can be achieved using many molecular
genetics techniques. The most prominent of
these techniques include:
PCR-based methods
Synthetic gene method
PCR and Restriction-free Cloning
Isothermal Assembly
16. Traditional PCR
When PCR is used for site-directed mutagenesis, the
primers are designed to include the desired change, which
could be base substitution, addition, or deletion. During
PCR, the mutation is incorporated into the amplicon,
replacing the original sequence.
Substituting bases in a sequence
18. PCR for Deletions Primer A contains
complementary
sequence to the
regions flanking the
area to be deleted.
During PCR, primer
binding will cause a
region of the
template to loop out,
and amplify only the
complementary
region. The final
product is shorter
because it is missing
the deleted
sequence.
19. PCR for Terminal Additions
Primer containing
an addition to the
sequence on the
5’ end (the 6X His
tag, primer B) is
used along with the
complementary
primer A to amplify
a new product
containing the
terminal addition.
Limitation!
While PCR for substitutions, additions, and deletions is a simple way
to introduce a mutation, it is limited by the fact that the mutation can
only be introduced in the sequence covered by the primers rather
than the sequence that lies between the primers
20. Primer extension
This can also be used for additions
and deletions of sequences. It
involves incorporating mutagenic
primers in independent, nested
PCRs to ultimately combine them in
the final product. The reaction uses
flanking primers (primers A and D)
on either end of the target sequence,
plus two internal primers (primers
B and C) that contain the
mismatched or inserted bases and
hybridize to the region where the
mutation will occur. The first round
of PCR creates the AB and CD
fragments. The two PCR products
are mixed together for a second
round of PCR. Because primers B
and C have complementary ends,
the two fragments will hybridize in
the second PCR with primers A and
D. The final product AD will contain
the mutated sequence.
Primer Extension for an Insertion
21. Inverse PCR
While traditional PCR amplifies a region of known
sequence, inverse PCR uses primers oriented in the
reverse direction to amplify a region of unknown
sequence. Mutagenic
primers can be used to change cloned sequences using a
technique adapted from the inverse PCR method. In this
method, the entire circular plasmid is amplified and a
sequence is deleted, changed, or inserted. The primers
are positioned ‘back-to-back’, facing outward, on the
two opposite DNA strands. One or both of the primers
contain the mismatches to create the desired mutations,
and both may also carry phosphorylated 5’ ends or a
restriction site for subsequent recircularization.
23. Synthetic Gene Method
Arguably, the most significant improvement to mutagenesis
methods is the commercial availability of long, synthetic,
double-stranded (ds), custom DNA fragments. Up to 3 kb
dsDNAs can be obtained with the desired mutations designed
directly into the sequences. They are compatible with both
existing methods (e.g., PCR, and restriction cloning), and new
methods (e.g., isothermal assembly) of mutagenesis, and are
becoming a standard reagent in these techniques because
they eliminate some of the time-consuming steps needed to
produce both a wild-type sequence and any derivative
variants. Now, for a reasonable cost, researchers can design
all the requisite sequences for their experiments, order them
online, and receive them ready for direct use or cloning.
Moreover, final constructs can be easily generated even when
a physical starting template is not available.
24. PCR and Restriction-free (RF) Cloning
dsDNA gene fragments are compatible with familiar PCR
mutagenesis methods, but they also offer some interesting
advantages. An example of the direct application of dsDNA to
PCR mutagenesis is the restriction-free (RF) cloning method.
In the RF method, PCR primers are replaced with long
dsDNA that has 5’ ends containing homologous overlaps with
the desired vector insertion site
25. Isothermal Assembly Isothermal mechanisms assemble
pieces of linearized DNA—
typically, a plasmid and one or
more inserts—with overlapping
homologous ends by first
modifying the DNA and then
joining the fragments. Typically,
the fragments are mixed together
in the reaction, and overhanging
ends are created by an enzyme
with endonuclease activity. The
resulting “sticky” ends then
anneal to the complementary
fragments, which determines the
precise position and directionality
of each piece in the finished
construct. A polymerase then fills
in the gaps, and a ligase seals the
nicks
26. Gene Mutation
Side-directed Mutagenesis
Mutagenic Agents and Consequences of Mutation
Epigenetics
Genetic Recombination
Genetic Diseases and Gene Therapy
Cell Cycle (phases and biochemical checkpoints)
27. Mutagenic Agents and Consequences of Mutation
Two important sources of mutations are inaccuracy in DNA
replication and chemical damage to the genetic material.
Mutagenic agents (also called mutagens) are chemical
substances that artificially induce mutations. They may be
grouped into physical and chemical mutagens.
28. The principal Physical mutagens are ionizing radiations which
cause mutations by producing free radicals which react with DNA
by forming dimers between adjacent thymine residues on the
same DNA strand, which may stop DNA synthesis.
29. Chemical mutagens are generally carcinogenic substances and
may be alkylating agents (which react with the DNA by
alkylating the phosphate groups as well as the purines and
Pyrimidines) or base analogs and intercalating agents.
30. Base analogs are structurally similar to proper bases and
therefore substitute for the normal bases cause errors in
replication. They base-pair inaccurately, leading to frequent
mistakes during the replication process. One of the most
mutagenic base analogs is 5-bromouracil, an analog of
thymine. The presence of the bromo substituent allows the
base to mispair with guanine via the enol tautomer.
31. Intercalating agents are flat molecules containing several
polycyclic rings that bind to the purine or pyrimidine bases of
DNA. They slip between the bases to cause deletion or addition
of a base pair or even a few base pairs. By slipping between the
bases in the template strand, they either cause the DNA
polymerase to insert an extra nucleotide opposite the
intercalated molecule or cause the polymerase to skip a
nucleotide. They include ethidium, proflavin and acridine
orange.
32. Determining the Sequence of Amino Acid
Residues
The Function of a Protein Depends on Its
Amino Acid Sequence
33.
34. The two major direct methods of
protein sequencing:
Edman degradation reaction (unknown
protein)
mass spectrometry
36. Mass spectroscopy Workflow
Protein sequence
IFRTKHKLDFTPIGCDAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTGESGMIVFRLLTKNNRWTWVQSNARLLYKNGRPDYIIVTQ
Trypsin digest
IFRTKHK
LDFTPIGCDAKGR
IVLGYTEAELCTRGSGYQFIHAADMLYCAESHIR
MIKTGESGMIVFRLLTK
NNRWTWVQSNARLLYK
NGRPDYIIVTQ
Mass spectroscopy
sample
time of flight
Peptide mass fingerprint
HKLDFTPIGCDAKGRIVLGYTEAELCTR
LLTKNNRWTWVQSNARLLYKNGR
TGESGMIVFR PDYIIVTQ
Mass Spectrometry
37. Protein Identification
2D-GE + MALDI-MS
– Peptide Mass Fingerprinting (PMF)
2D-GE + MS-MS
– MS Peptide Sequencing/Fragment Ion Searching
Multidimensional LC + MS-MS
– MudPIT (Multidimensional Protein Ident. Tech.)
1D-GE + LC + MS-MS
Matrix-assisted laser desorption ionization
MALDI:
39. Amino Acid Sequences Provide
Important Biochemical Information
Knowledge of the sequence of amino acids
in a protein can offer insights into its three-
dimensional structure(prediction) and its
function, cellular location, and evolution.
40. Protein Sequence Comparison
Compare primary sequence of homologous protein,
We will find:
invariant residues or conserved residues
This residues are important for function and structure
53. Secondary Structure
Three main
–α - helix
– β- sheet
– β- turn
• Driving force for the formation of secondary
structure is the formation of H-bonds in the
peptide backbone
“Local structures”
54. Alpha-Helix
• First proposed by Linus Pauling and
Robert Corey in 1951
• A ubiquitous component of proteins
• Stabilized by H-bonds
55. Alpha-Helix
•Residues per turn: 3.6
•Rise per residue: 0.15
nm
•Rise per turn (pitch):
3.6 x 0.15nm = 0.54nm
Right handed
helix
The α helix is a right-
handed spiral
The Alpha-Helix is a rigid, rodlike structure
56. Hydrogen Bond Pattern in α Helix
The α helix is stabilized by extensive hydrogen
bonds
amino hydrogen H-bonds with carbonyl oxygen
located 4 AA’s away forms 13 atoms loop
57. Alpha-Helix
•Side chain groups point
outwards from the helix
•AA’s with bulky side chains
less common in alpha-helix
•Glycine and proline
destabilizes alpha-helix
•So not in alpha-helix structure
Pro has no N-H group available to form intrachain hydrogen bonds
58. Beta-Sheets
Beta-sheets formed
from multiple side-
by-side β-strands
stabilized by
hydrogen bonds
that form between
the polypeptide
backbone H-atom
and carbonyl
groups of adjacent
chains
59. Beta-Sheets
Can be in parallel
or antiparallel
configuration
Antiparallel beta-
sheets more
stable
The Beta-Sheet is a rigid
61. The random coil is not a true secondary
structure, but is the class of conformations
that indicate an absence of regular
secondary structure.
Functional regions in
enzyme structure
so flexible
62. Many globular proteins contain combinations
of Alpha-Helix and Beta-Sheets secondary
structure. These pattern are called
supersecondary structure.
Supersecondary structures, also called motifs ,
are particularly stable arrangements of several
elements of secondary structure and the
connections between them.
63.
64.
65.
66.
67. Many proteins are composed of several discrete,
independently folded, compact units called domains.
Domains may consist of combinations of motifs. The
size of a domain varies from as few as 25 to 30 amino
acid residues to more than 300.
Note that each domain is a distinct compact unit
consisting of various elements of secondary structure.
68.
69. Polypeptides with more than a few hundred amino acid residues
often fold into two or more stable, globular units called domains.
70.
71. Tertiary Structure
Globular proteins have a variety of tertiary structures
Tertiary structure is concerned with the arrangement in
space of all atoms in a polypeptide chain
The formation of the 3°structure is primarily
determined by the interactions of the amino acid side
chains with each other and the backbone atoms
74. Quaternary Structure of Proteins
Many proteins consist of more than one
polypeptidechain
Subunits - different polypeptide chains
The individual subunits associate in a
specific geometry
for that protein known as the quaternary
structure Subunits interact via non-
covalent interactions
77. Subunits - different polypeptide chains
• Proteins with more than one subunit are called oligomers
– Dimer, trimer, tetramer etc.
Subunit
78. amino acid sequence secondary
structure
supersecondary
structure
structural domain & tertiary
structure
quaternary structure
Different Levels of Protein Structure
79. Summary
1. Proteins are made from 20 standard amino acids each of which
contains an amino group, a carboxyl group, and a side chain, or R
group. Except for Gly, which has no chiral carbon, all amino acids
in proteins are of the L configuration.
2. The side chains of amino acids can be classified as having
highly hydrophobic or highly hydrophilic side chains on the basis
of the polarity and charge (at pH 7) of their R groups.
3. The properties of the side chains of amino acids are important
determinants of protein structure and function. The charges of
ionizable side chains depend on both the pH and their pKa values.
80. 4. There are four levels of protein structure: primary (sequence
of amino acid residues), secondary (regular local conformation,
stabilized by hydrogen bonds), tertiary (compacted shape of
the entire polypeptide chain), and quaternary (assembly of two
or more polypeptide chains into a multisubunit protein).
5. Amino acid residues in proteins are linked by peptide bonds. The sequence
of residues is called the primary structure of the protein.
6. Secondary structure is the local spatial arrangement of the
main-chain atoms in a selected segment of a polypeptide chain.
The most common regular secondary structures are the helix,
the conformation, and turns. hydrogen-bonded to each other
to form b sheets
81. 7.Tertiary structure is the complete three-dimensional structure of a
polypeptide chain. There are two general classes of proteins based
on tertiary structure: fibrous and globular.which serve mainly
structural roles, have simple repeating elements of secondary
structure.
8. Globular proteins have more complicated tertiary structures, often
containing several types of secondary structure in the same
polypeptide chain.
9. Quaternary structure results from interactions between the subunits
of multisubunit (multimeric) proteins or large protein assemblies. In
proteins that possess quaternary structure, subunits are usually held
together by noncovalent interactions.
82. 10. Proteins with very similar amino acid sequences are
homologous—they descend from a common ancestor.
11. A comparison of sequences from different species
reveals evolutionary relationships.
83. Reference Text Books
David L. Nelson and Michael M. Cox
LEHNINGER PRINCIPLES OF BIOCHEMISTRY
(Fifth Edition)
Moran. Horton, Scrimgeous.Perry
PRINCIPLES OF BIOCHEMISTRY