This document discusses two molecular marker techniques: RAPD and RFLP. RAPD (Random Amplified Polymorphic DNA) is a PCR-based technique that uses short arbitrary primers to detect variations between individuals' genomes. RFLP (Restriction Fragment Length Polymorphism) is a hybridization-based technique that involves restriction enzyme digestion of DNA, gel electrophoresis to separate fragments, Southern blotting, hybridization with probes, and autoradiography to detect variations in fragment lengths between individuals. Both techniques are useful for genetic mapping, trait mapping, phylogenetic analysis, and DNA fingerprinting.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
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.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
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.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Introduction to RDT methods in genetic engineeringCollege
This presentation gives a small review about the RDT based methods used generally in genetic engineering. This presentation include various images about the technique, which will help the user in understanding the concepts easily.
DNA Fingerprinting & its techniques by Shiv Kalia (M.Pharma in Analytical Che...Shiv Kalia
DNA fingerprinting and below mention content widely cover in this presentation
History & Introduction of DNA fingerprinting
How was the first DNA fingerprint produced?
Types of DNA Based Markers
Polymerase Chain Reaction (PCR)
PCR based Methodology of DNA fingerprinting
Electrophoresis
Utility of DNA Based Markers
Various DNA Fingerprinting Techniques Advantages & Disadvantages
Authentication of Various Ayurvedic Herbs by DNA Fingerprinting
Advantages of DNA fingerprinting in Plants
Disadvantages of DNA fingerprinting in Plants
CONCLUSION
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
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Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
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 .
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.
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/
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
(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. Molecular markers:
The variant DNA fragment that
gives some information about genes of
interest is called molecular
marker/genetic marker.
3. Types of molecular marker:
1.PCR based marker.
RAPD.
2.Hybridization based markers.
RFLP.
AFLP.
SNP.
4. Restriction fragment length polymorphism:
The variation in the restriction DNA
fragment lengths between individuals of a
species is called restriction fragment length
polymorphism. This is a best laboratory
technique to analyze and compare DNAs of two
or more individuals of a species. It is
extensively used in genome analysis.
5. RFLP method involves the following steps:
Sample collection.
Isolation DNA.
Restriction digestion.
Electrophoresis.
Blotting of DNA.
Making genomic DNA probes.
Nucleic acid hybridization.
Autoradipgraphy.
6. sampe collection:
Tissues or cells of individuals are collected to extract their
DNA. The samples are collected separately.
Isolation of DNA:
The DNA was isolated from the tissues or cells.
restricton digestion:
Genomic DNA of each sample is cut with a restriction
enzymes separately to generate variable lengths of DNA
fragments.restricion enzymes such as EcoRI .Hind III, PstI are of
much use for RFLP. This restriction digest is divided into two halves
one half is used for DNA DETECTION and other is used for PROBE
MAKING.
7. Electrophoresis:
The digested genomic DNA of all the samples are loaded
into separate wells in agarose or polyacrylamide gel and are
subjected to electrophoresis. The DNA fragments get separated
according to their size .use of molecular weight markers in one lane
is likely to know the molecular weight of separated DNA fragments.
Blotting of DNA :
As in southern blotting the DNA is transferred to a
nitrocellulose filter by using a blotting setup. The nitrocellulose filter
is dried in between filter discs before performing hybridization.
8. Making genomic DNA probes:
One half genomic DNA digest from each sample is
electrophoresed to separate the DNA fragments. Fragments of 0.5-
2.0 kb are extracted from the gel and cloned in Puc21 vector to
construct rDNAs.
These rDNA are amplified by introducing them into bacterial
host and the amplified rDNAare isolated from the bacteria . the
target DNA fragments are excised from the rDNA by using the
same restriction enzyme that was Used for cutting the DNA. They
are purified by electrophoresis.
9. These DNA fragments are radiolabelled with p35 isotope
incorporated nucleotide by nick translation or random primer
extension or end labeling. As a result, genomic probes are
formed
Nucleic acid hybridization:
DNA blotted nitrocellulose membrane is kept immersed
in a hybridization solution containing genomic DNA probes to
bring out hybridization. After hybridization unbound probes are
washed out of the membrane.
10. Autoradiography:
Images of radioactive probes are captured on an X-ray
film using autoradiography. The autoradiogram shows DNA
bands in distinct lanes each of which is characteristics of an
individual. Variation in the lengths of DNA fragments between
individuals as shown by the lanes in the autoradiogram is called
RFLP.
13. Random amplified polymorphic DNA :
The RAPD is a PCR based method to detect
variations between individuals of a species by
selective amplification of some polymorphic
sequences in their genome. this method was
developed by J.G.K.WILLIAMS in 1991. only
least number of DNA fragments are considered for
RAPD analysis. RAPD are of much use to
construct genetic maps.
14. Steps involved in RAPD analysis:
1.Sample cells or tissues are collected from individuals to be
distinguished from one another.
2.Genomic DNA of each and every sample is isolated by using a
standard procedure.
3.each genomic DNA is separately treated with Taq polymerase, a
primer ,Datp,dttp,dctp,dgtp.the primer is 9-10 bp
4. all these reaction mixture are subjected to repeated cycles.
15. denaturation,primer annealing, polymerization upto 35-40 cycles.
Denaturation: temperature is maintained at 940 c for 1 mins
Annealing:360c for 2 mins
Polymerization: 720c for 1.5 mins
Repeated cycle of denaturation, annealing, polymerization leads to
amplification of polymorphic sequences in the genome.
5.As a result of PCR amplification each reaction tube contains RAPD
fragments. Ethidium bromide is added to it.
6.The RAPDs thus obtained from the samples are separately loaded
into wells of an AGE or PAGE and molecular weight markers are
added on the well.
16. 7.The loaded DNAs are electrophoresed to separate the DNA
fragments based on their size.
8.The electrophoresed gel is examined under a UV light illuminator to
detect light bands and photographed.
If a DNA band of molecular weight 1Kb distinguish an
individual from others it is considered as RAPD marker of that
individual. An individual may differ from others in one or more
RAPD markers.
17.
18. Advantages:
There is no need for species specific probes in RAPD analysis.
RAPD is quick method.
RAPD analysis can be performed in crude DNA samples also.
RAPD requires only small amount of DNA samples.
It does not require radioactive probes and hybridization.