This document discusses various techniques for DNA sequencing and analysis. It begins by explaining how electrophoresis can separate DNA molecules based on size. It then describes Sanger dideoxy sequencing, which uses chain-terminating inhibitors to generate truncated DNA fragments that are separated by electrophoresis. Next, it discusses DNA footprinting to identify regulatory sequences and single-strand conformation polymorphism analysis to detect mutations. The document provides details on these various DNA sequencing and analysis strategies.
Amplification techniques used in molecular biology for amplifying a target DNA to produce millions of copy.
An important tool in molecular study and analysis.
Amplification techniques used in molecular biology for amplifying a target DNA to produce millions of copy.
An important tool in molecular study and analysis.
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by transduction (Bitner, Kuempel 1981).
Restriction mapping is a useful way to characterise a particular DNA molecule. It enables us to locate and isolate DNA fragments for further study and manipulation. The relative location of different restriction enzyme sites to each other are determined by enzymatic digest of the DNA with different restriction enzymes, alone and in various combinations.The digested DNA is separated by gel electrophoresis and the fragment sizes that have been generated are used to build the 'map' of sites of the fragment. The map lets us know 'where we are' in the linear DNA macromolecule.
Gene mapping | Genetic map | Physical Map | DNA Data Analysis (upgraded)NARC, Islamabad
Genes are useful markers but not ideal.
Mapped feature that are not genes are called DNA markers.
DNA markers must have at least two alleles to be useful.
DNA sequence features that satisfy this requirement are-
– Restriction Fragment Length Polymorphism (RFLP)
Southern hybridization
PCR
– Simple Sequence Length Polymorphism (SSLP)
– Single Nucleotide Polymorphism (SNP)
Mapping- determining the location of elements with in a genome, with respect to identifiable land marks.
Gene mapping describes the methods used to identify the locus of a gene and the distances between genes.
In simple mapping of genes to specific locations on chromosomes.
Two types
Genetic map
Physical Map
They are useful in predicting results of dihybrid and trihybrid crosses.
It allows geneticists to understand the overall complexity and genetic organization of a particular species.
Identify genes responsible for diseases.
Identify genes responsible for traits.
genetic maps are useful from an evolutionary point of view.
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by transduction (Bitner, Kuempel 1981).
Restriction mapping is a useful way to characterise a particular DNA molecule. It enables us to locate and isolate DNA fragments for further study and manipulation. The relative location of different restriction enzyme sites to each other are determined by enzymatic digest of the DNA with different restriction enzymes, alone and in various combinations.The digested DNA is separated by gel electrophoresis and the fragment sizes that have been generated are used to build the 'map' of sites of the fragment. The map lets us know 'where we are' in the linear DNA macromolecule.
Gene mapping | Genetic map | Physical Map | DNA Data Analysis (upgraded)NARC, Islamabad
Genes are useful markers but not ideal.
Mapped feature that are not genes are called DNA markers.
DNA markers must have at least two alleles to be useful.
DNA sequence features that satisfy this requirement are-
– Restriction Fragment Length Polymorphism (RFLP)
Southern hybridization
PCR
– Simple Sequence Length Polymorphism (SSLP)
– Single Nucleotide Polymorphism (SNP)
Mapping- determining the location of elements with in a genome, with respect to identifiable land marks.
Gene mapping describes the methods used to identify the locus of a gene and the distances between genes.
In simple mapping of genes to specific locations on chromosomes.
Two types
Genetic map
Physical Map
They are useful in predicting results of dihybrid and trihybrid crosses.
It allows geneticists to understand the overall complexity and genetic organization of a particular species.
Identify genes responsible for diseases.
Identify genes responsible for traits.
genetic maps are useful from an evolutionary point of view.
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA.
You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.
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.
Richard's entangled aventures in wonderlandRichard 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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
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.
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 .
(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.
2. INTRODUCTION
DNA is a long rod-like molecule which moves
through polyacrylamide gels with limited mobility.
Moreover, DNA has a uniform charge-distribution
which means that this mobility is directly
proportional to the size of the molecule.
For these reasons, electrophoresis of DNA in
polyacrylamide gels allows separation of molecules
differing by as little as a single nucleotide
3. SANGER DIDEOXYNUCLEOTIDE SEQUENCING OF
DNA
SEQUENCING OF DNA
FOOTPRINTING OF DNA
SINGLE STRAND CONFORMATION POLYMORPHISM
ANALYSIS OF DNA
SEQUENCING STRATEGIES
FOR THE STUDY OF DNA
4. SANGER DIDEOXYNUCLEOTIDE SEQUENCING OF
DNA
In Sanger dideoxynucleotide sequencing, the
sequence of a single DNA strand is determined
In order to obtain large amounts of single stranded
DNA the M13 bacteriophage system is used.
5. DNA to be sequenced is cloned into the
RF form of M13.
This is transformed into competent lac-
E. coli and replicated first into several
copies of RF M13 and then into many
copies of single stranded Ml3.
Coat proteins surround this DNA to form
the mature bacteriophage which is
released into the medium.
Single stranded DNA is collected by
phenol-chloroform extraction and
ethanol precipitation.
This is used as template for DNA
6. A small amount of competitive
inhibitors of DNA polymerase,
dideoxyadenine, dideoxyguanine,
dideoxythymidine or
dideoxycytosine (ddNTPs) is
included in each of four separate
reactions, together with
substrates for this enzyme; dATP,
dGTP, dCTP and dTTP
The experiment therefore
results in four sets of DNA
daughter-strands which are
truncated fragments
complementary to the DNA to
be sequenced.
7. SEQUENCING OF DNA
The four mixtures are then electrophoresed in an
unusually long (up to 48 cm) and thin (0.4 mm)
continuous polyacrylamide (4–6%) gels in the
presence of urea (8 M) and at approx. 30–40 ◦C
A shark’s tooth comb is used to create small
reservoirs or wells at the top of the gel.
These form between the teeth of the comb and the
gel surface
8. Truncated fragments for each of
four chain termination reactions
are separated in polyacrylamide
gels containing 8 M urea.
Smaller fragments electrophorese
furthest into the gel (for clarity,
only those for ddG are shown in
the illustration but bands in the C,
A and T lanes arise from
corresponding fragment sets for
ddC, ddA and ddT).
Bands are visualised by
autoradiography on X-ray film.
The sequence obtained is
complementary to the sequence
AUTORADIOGRAM OF DNA SEQUENCING GEL
9. Primer walking approach for sequencing 1 kb DNA
fragment. New oligonucleotide primers are designed
from 3 ends of previously sequenced region. In this
way sequencing of a template can be continued until
completed.
10. Automated DNA sequencing.
Instead of a radiolabel, primer
strands are labeled with a
different fluor or each ddNTP.
These have distinct excitation
(λ1) and emission (λ2)
wavelengths and can
therefore be easily
distinguished by the detector.
A single track can be used for
separation of all four ddNTP
nested fragments.
AUTOMATED DNA
SEQUENCING.
11. FOOTPRINTING OF DNA
In eucaryotes, most DNA sequences are noncoding,
that is they do not code for protein
Examples include satellite DNA, intervening
sequences within genes (introns) and regulatory
sequences.
A widely-used experiment allowing the identification
of regulatory DNA sequences is DNA footprinting
12. This experiment involves mixing genomic DNA (or
smaller fragments derived from genomic DNA) with an
extract containing the protein factor under
investigation followed by digestion with a small
amount of DNA-ase I.
13. An adaptation of DNA footprinting allows simultaneous
identification and sequencing of the regulatory site
14. SINGLE STRAND CONFORMATION
POLYMORPHISM ANALYSIS OF DNA
widely-used screening method for the identification of
mutations in DNA called single strand conformation
polymorphism (SSCP) analysis
The procedure involves heating DNA fragments (200– 400 bp)
of a single gene from a range of samples (e.g. different
human individuals) in either NaOH or formamide to denature
double-stranded DNA.
This technique has come to be widely-used in the screening
of human DNA for mutations responsible for genetically-
based diseases.
15. (a)DNA is denatured to make it single-
stranded. When placed in non-
denaturing conditions, intrachain
hydrogen bonding can occur.
Mutations in single-stranded DNA
may alter the intrachain hydrogen
bonding pattern giving a
conformational polymorphism
(b) Wild-type and mutant DNA have
altered mobilities in fragments
containing mutations. These are
visualised as band-shifts in
polyacrylamide non-denaturing gels.
Bands can either be shifted up (sample
2) or down (sample 3).