This document discusses various molecular methods used in molecular genetics and molecular biology. It begins by categorizing common techniques into diagnostic methods, analysis and sequencing, microarray methods, and proteomics. Several key diagnostic techniques are then described in more detail, including gel electrophoresis, hybridization, PCR, immunoprecipitation, and karyotyping. Recombinant DNA technology basics like cloning genes, cDNA, and purposes of cloning are explained. Other analysis methods such as sequencing, microarrays, RNA-Seq, and techniques like RNAi, knockout methods, and liposomes are also summarized.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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Probes are used for hybridization purposes. different types of probes can be used on the basis of what we want to hybridize. May be Radioactive or Non-Radioactive.
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
https://www.patreon.com/biotechlive
SUPPORT EDUCATION... SUPPORT US
Probes are used for hybridization purposes. different types of probes can be used on the basis of what we want to hybridize. May be Radioactive or Non-Radioactive.
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
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.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
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.
Biology Molecular
University of Haifa. (2015, August 13). Big data and the social character of genes. ScienceDaily. Retrieved September 12, 2015 from www.sciencedaily.com/releases/2015/08/150813084035.htm
Lawrence Berkeley National Laboratory. (2015, August 4). Keeping algae from stressing out. ScienceDaily. Retrieved September 12, 2015 from www.sciencedaily.com/releases/2015/08/150804144013.htm
Martínez Sánchez, Lina María. Biología Molecular. 8.ed. Medellín. UPB. Facultad de Medicina.
Southern blotting is a laboratory technique used to detect specific DNA sequences in DNA samples. It involves several steps:
Restriction Digestion: DNA from a biological sample (such as blood or tissue) is broken into smaller fragments using restriction enzymes, which cut the DNA at specific sequences.
Electrophoresis: The DNA fragments are separated based on their molecular weights using gel electrophoresis. This process allows smaller fragments to move faster than larger fragments.
Transfer to Membrane: The DNA fragments are transferred from the gel onto a solid membrane, typically a positively charged nylon membrane, using capillary action.
Hybridization: The membrane is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The probe is designed to be complementary to the target DNA sequence, allowing it to bind to the specific DNA fragment on the membrane.
Detection: The bound probe is detected using methods such as X-ray film, phosphorimaging, or chemiluminescent substrates, depending on the type of label used.
Southern blotting is used in various applications, including:
Identifying specific DNA sequences in DNA samples
Studying gene rearrangements and mutations
Analyzing viral and bacterial infections
Forensic analysis and personal identification
Gene mapping and restriction enzyme mapping
Identifying methylated sites in genes.
This technique is named after its inventor, Dr. Edwin Southern, who first published it in 1975.
This is all about southern blotting
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
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.
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.
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.
(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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
2. Molecular biology techniques are common
methods used in molecular biology, biochemistry,
genetics, and biophysics which generally involve
manipulation and analysis of DNA , RNA, Protein,
and Lipid.
6. Why is it necessary to clone genes?
• Naturally occurring DNA molecules are very long
and a single molecule usually carrying many
genes
• Genes may occupy only a small proportion of the
chromosomal DNA and the rest are noncoding
sequence (a human gene might constitute only
1/1,000,000 of a chromosomal DNA)
7. Making cDNA
from an
Eukaryotic Gene
• To express an
eukaryotic gene into
its product in bacterial
cells, the mRNA is
converted into cDNA
• The cloned cDNA can
be expressed into
protein product by
inserting it into a
plasmid containing a
functional promoter
(expression vector)
8. Purposes of Cloning Genomic DNA and cDNA
• Genomic DNA:
Studying structures of genes, gene families
Identifying promoters and other regulatory elements
Studying evolution of genes
• cDNA:
Studying structures of mRNA
Measuring the levels of mRNA
Studying developmental stage-specific and tissue-specific
expression of genes
Studying processing and stability of mRNA
Producing recombinant proteins
9. • Detection of Nucleic Acids
Agarose gel electrophoresis
Hybridization, Southern blot and RNA
northern blot analyses
10. Agarose Gel Electrophoresis to
Separate DNA
• Agarose is an inert carbohydrate isolated from seaweeds. DNA
molecules of different sizes can be separated in an agarose gel by
electrophoresis and visualized by staining the gel with ethedium
bromide & observe under UV light. The DNA fragment can be
recovered from the gel by extraction with phenol and chloroform
11. DNA Fragments Visualized Under UV Light
DNA on agarose gel
is stained with
ethedium bromide
and observed under
UV light
12. Hybridization
• Hybridization: Two fragments of single-stranded
homologous DNA molecules can form hybrid through
hydrogen-bonding formation (base pairing)
5’-GTACTTAGGCAATTGGGCA-3’
3’-CATGAATCCGTTAACCCGT-5’
• If one of these two strands of DNA is labeled with
radioactive isotopes, the hybrid will be easily visualized
by autoradiography
• Hybridization can occur between two homologous DNA
molecules or a DNA molecule and a RNA molecule
• Southern blot and RNA northern blot hybridization
16. Fluoerescence in situ Hybridization
This technique is used for cytological localization of molecules in the cell
17. Colony
Hybridization
• This method is
based on the
principle that two
homologous
strands of nucleic
acids can form
hybrid form
• If one of the
strands of nucleic
acid is radio-
labeled, the hybrid
can be visualized
by
autoradiography
18. A Single Strand DNA to be Sequenced
Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980
23. Chromosome
Walking
• This technique
allows the
isolation of a long
eukaryotic gene
• An alternative is to
construct a BAC
library that
contains long
piece DNA
molecules
25. Nuclear Run-on
Transcription Assay
• Nuclear run-on assay can be
used to ascertain which gene is
active in a given cell allowing
transcription to continue in
isolated nuclei
• Specific transcript can be
identified by their hybridization
to known DNAs on dot blot
• It can also be used to determine
the effects of assay conditions
on nuclear transcription
• Transcription activity of a
specific gene can be determined
• It can also be used to measure
template activity
26. Nuclear Run-On Assay to Measure the
Transcription Rates of Genes in Various Tissues
28. • technique that allows you to
determine the expression of many
genes at one time
29. Principle of DNA
Microarray
• Genes (cDNA or
oligonucleotides are
spotted on glass
plates
• Messenger RNA is
reverse transcribed
into cDNA and labelled
with Cy3 (emission
570nm, green) or Cy5
(emission 670 nm, red)
• Hybridization (mixing
Cy3-cDNA and Cy5
cDNA)
• Scan the slide to
detect the
hybridization signals
30. APPLICATIONS
• Gene discovery: Examples
Profiling of cancer-specific expressing genes
Tissue-specific expression of genes
Developmental-specific expression of genes
• Disease diagnosis:
Collections of genes showing expression of genes specific
to certain types of diseases
Examples: Specific cancer type, hematopoietic disease etc.
• Drug discovery: Pharmacogenomics
To find correlations between therapeutic responses to drug
and gene profiles of patents
• Toxicological research: Toxicogenomics
• To find correlations between toxic responses to toxicants
and chages in the gene profiles of the objects exposed to
such toxicants
31. Enlarged Photo of a Microarray Chip
• This array has
2400 human
genes
• Red indicates
increase of
expression;
yellow equal
expression and
green reduce of
expression
• This technique
can help to
determine the
profiles of gene
expression
33. RNA-seq, also called "Whole Transcriptome Shotgun
Sequencing" ("WTSS"), refers to the use of high-
throughput technologies to sequence cDNA or RNA in
order to get information about a sample's RNA content.
The technique has been adopted in studies of diseases
like cancers. With deep coverage and base-level
resolution, this technology provides information on
differential expression of genes, including gene alleles
and differently spliced transcripts; non-coding RNAs;
post-transcriptional mutations or editing; and gene
fusions
37. • IP is the technique of precipitating a
protein antigen out of solution using
an antibody that specifically binds to that
particular protein.
• This process can be used to isolate and
concentrate a particular protein from a
sample containing many thousands of
different proteins.
• Immunoprecipitation requires that the
antibody be coupled to a solid substrate at
some point in the procedure.
38.
39. KARYOTYPING
• Is the study of the structure and properties of
chromosomes, chromosomal behaviour during
mitosis and meiosis, chromosomal influence on
the phenotype and the factors that cause
chromosomal changes.
42. KARYOTYPE PREPARATION
• Cultured cells are arrested at metaphase by
adding colchicine
• This is when cells are more condensed and
easiest to identify
• Arrested cells are broken up
43. • Metaphase chromosomes are fixed and stained.
• Chromosomes are photographed through microscope
Photograph of chromosome is cut up and arranged to
form karyotype diagram.
46. • Technique that is used in genetic screening to identify
specific areas of interest in the genome , and then
determine what they do.
• Genes related to conditions such as Huntington's
Disease and cystic fibrosis have been identified with
this technique.
47. Positional Cloning- identification the cystic
fibrosis (CF) gene:
Most common lethal genetic disease in the U.S. (~1 in
2,000).
First human gene identified by positional cloning.
Required 4 years and the work of many laboratories.
48. OVERVIEW OF CYSTIC
FIBROSIS:
CF results from defect in protein that regulates
the movement of salt and water in and out of
cells.
Causes thick mucus secretions in the lungs,
pancreas, and intestines.
Causes lung disease and organ failure, patients
experience chronic bacterial infections.
Life expectancy is abut 40 years.
50. • Liposomes are spherical, self-closed structures
formed by one or several concentric lipid bilayers
with an aqueous phase inside and between the lipid
bilayers.
• Their ability to entrap different water-soluble
compounds within the inner aqueous phase and
lipophilic agents between liposomal bilayers has
made them useful for delivery of different kinds of
drugs.
51. • Liposomal modification with (PEG) increases their
field of usage by enhancing circulation time and
attachment of antibodies or different targeting
moieties to their surface to targe.
• considered for intravascular drug delivery, using
cells and noncellular components as the targeted sites
for diagnosing and treating the most important
cardiac pathologies, including myocardial infarction,
coronary thrombosis, and atherosclerosis at specific
affected areas.
53. DNA is denatured into single strands
annealed to random hexamer oligonucleotides.
These random primers can then be extended using
Klenow enzyme
Random pieces of DNA for can be used for probe
production.
These probes can be used on blots or DNA
microarrays.
