Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Southern Blotting (SB) 4 jan 2015 finalICHHA PURAK
The power Point presentation contains 38 slides explaining about different steps involved in Southern Blotting such as DNA Isolation, Restriction digestion, Separation of DNA fragments by gel electrophoresis, denaturation of Double stranded DNA , transfer of fragments from gel to membrane ( blotting) , hybridization and detection by autoradiography. Applications of Southern blotting have also been discussed
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
Southern Blotting (SB) 4 jan 2015 finalICHHA PURAK
The power Point presentation contains 38 slides explaining about different steps involved in Southern Blotting such as DNA Isolation, Restriction digestion, Separation of DNA fragments by gel electrophoresis, denaturation of Double stranded DNA , transfer of fragments from gel to membrane ( blotting) , hybridization and detection by autoradiography. Applications of Southern blotting have also been discussed
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
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.
FAIRSpectra - Towards a common data file format for SIMS imagesAlex Henderson
Presentation from the 101st IUVSTA Workshop on High performance SIMS instrumentation and machine learning / artificial intelligence methods for complex data.
This presentation describes the issues relating to storing and sharing data from Secondary Ion Mass Spectrometry experiments, and some potential solutions.
Nutrition is the science that deals with the study of nutrients and their role in maintaining human health and well-being. It encompasses the various processes involved in the intake, absorption, and utilization of essential nutrients, such as carbohydrates, proteins, fats, vitamins, minerals, and water, by the human body.
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.
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.
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.
3. 1. Sexual assault & homicide unit
2. Paternity unit
3. Human identification unit
4. Mitochondrial dna unit
Profiling tools and techniques:
DNA Forensic Laboratory is estimated to increase the
capacity to examine to 2,000 cases per year
1. Disputed paternity/maternity
2. Criminal paternity
3. Rape and murder cases
4. Child abuse and sexual assault
cases (POCSO)
5. Human identification cases
6. Forensic challenged DNA cases:
charred bone , exhumed skeletal
remains, touch DNA, trace
elements etc.
7. Homicide cases
8. Kinship analysis.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. Liquid form
Crime scene
(24 hours)
Collect in EDTA tube using
syringe or dropper Or Transfer
on gauze piece / FTA card. Air
dry
Fresh / Wet
clot Crime
scene
sterile tube and add equal
volume of normal saline /
PBS Or Transfer on gauze
piece / FTA card. Air dry it
and keep in paper packet /
envelope with desiccant.
Wet / damp
Crime scene,
clothing,
fabrics, Victim’s
clothing,
suspect’s
clothing etc
Thoroughly air dry at room
temperature. Roll it in clean in
paper or brown paper
17. Wet blood
on
Object
Thoroughly air dry at room
temperature. Collect the item as
it is. Pack in paper bag /
envelope , cardboard / shipping
boxes, depending upon the size
of object
Crust / stain
/Spatters
Crime scene,
or Unmovable
surface, floor,
concrete wall
etc
Moistened the dry blood stain
for 5-10 minutes with PBS /
distilled water. Collect the
moistened stain with foam
tipped swab / FTA card / gauze
piece and air dry the swab.
Stain
Weapon/firearm
/bullet Small
objects such as
household
utensils, stones,
bricks etc.
Allow the stains to dry. Collect
the item directly
18. SEMEN
Stain Vehicle upholstery,
carpet, wallpaper, wood
etc.
**Cut out the stained
area. Allow it to dry in
shade. Also collect an
unstained cutting as a
control from adjacent area.
Liquid form Object, crime
scene
** Collect the sample with
sterile gauze piece /
cotton swab / surface
swab. Air dry the swab
Wet / semi dry Mutilated
remains at crime scene or
place of recovery
**** Immediately store
parcel under freezing
conditions without any
preservative for DNA
analysis.
19. Bones / teeth
Wet / semi dry / dry Crime scene
or place of recovery
Clean and wash the bones and
teeth to remove any debris.
Allow it to dry completely in air.
20. Hair with root Dry or wet with
blood, semen,
saliva, Crime scene,
weapon, clothing
Collect the sample
with help of
tweezers / forceps
in white paper /
butter paper and
pack in paper
envelope. If found
attached in dry
blood, weapon etc.
do not remove
21. The following items may contain DNA
material
This Photo by Unknown Author is licensed under CC BY
22. ACTIVITY
Clothings, Pubic and head hair, evidence from body,
swab/smear (genitals : vulva, cervix, vaginal), vaginal
wash, oral swab
Penile swab, nail clippings, urine sample
24. LABORATORY ORGANIZATION (Equipment's/sterilization and bleaching/PPE for analyst, storage and
analysis area seperation
WORK PLACE PREPARATION (sterilization with 10% bleach and/or 70% ethanol, gloves)
LABORATORY WASTE DISPOSAL MANAGEMENT (Human blood, other potential infectious body
fluid, Laboratory waste from infectious agents, Nucleic acid (natural & synthetic) containing
material, Sharps, Pipette tips)
SAMPLE HANDLING (DNA extraction and PCR in separate rooms, sterilization, sample handling
minimum, documentation.
BODY FLUID IDENTIFICATION ( in serious cases)
25. extraction negative
control
• 2pg/microlitre
PCR controls
• A positive control: DNA
sample where the known
STR alleles of loci
• A negative control
Parallel (Extraction/
amplification)
CONCORDANT
ANALYSES AND
“DUPLICATE RULE”
EXOGENEOUS
POLICY
DNA EXTRACTION GUIDELINES
26.
27. Forensic STR Analysis
DNA extraction
DNA quantitation
PCR amplification
(multiplex)
Electrophoresis
Interpretation of STR
profile
28.
29.
30. DNA EXTRACTION METHODOLOGIES
● reliable and efficient, it is also very time-consuming, uses hazardous chemicals, and, because of the
greater hands-on effort and multiple tube transfers involved, introduces increased opportunities for
contamination and sample mishandling
Phenol:chloroform:isoamyl alcohol (25:24:1)
ethanol precipitation or
through the use of a
centrifugal filter unit (i.e.,
Vivacon or Amicon devices),
31. Chelation Extraction
Chelex from Bio Rad (Fig.
21.4). Consisting of a
styrene divinylbenzene
copolymer containing paired
iminodiacetate ions
fast, can be easily automated, and require
minimal sample transfer.
inhibitory compounds (e.g., hematin and
immunoglobulin gamma in whole blood or
humic acid in soil-contaminated samples).
32. Solid Phase Extraction
More readily automatable extraction techniques
involve solid phase extraction methods
selectively bind DNA to a solid surface, such as silica in the presence of high
concentrations of chaotropic salts.
The bound nucleic acids and associated substrates are then separated from the
remaining cellular debris through the use of magnets or centrifugation.
Purified DNA is then readily eluted from the solid surface by the immersion of low
ionic strength or pH-adjusted buffers, allowing for nucleic acid recovery and
concentration.
33. QIAamp spin columns
QIAamp spin columns from
Qiagen selectively bind DNA to
silica in the presence of
chaotropic salts and elute DNA
under alkaline conditions
34. DNA IQ kit from the Promega Corporation
silica-based paramagnetic binding
resin that allows for the use of
magnets to isolate the silica-
bound DNA on the side of a
sample tube. This eliminates the
need for tube transfers during the
wash steps
35. The PrepFiler system by Thermo Fisher Scientific
https://www.thermofisher.com/in/en/home/industrial/forensics/human-identification/forensic-dna-
analysis/sample-preparation-extraction/prepfiler-forensic-dna-kits.html
paramagnetic resin,
but rather than
silica, it employs a
coating of a dextran
derivative, which
binds DNA in the
presence of alcohol
36. Differential Extraction
this is a pre-PCR approach that physically isolates
male cells for autosomal STR analysis, which offers a
superior power of discrimination.
This is in contrast to the use of Y-STRs, which simply
use male targeted genetic markers to selectively
amplify male DNA in the presence of excess female
DNA.
37.
38. ISOLATION OF DNA FROM BLOOD/ SALIVA/
OTHER BODY FLUIDS ARCHIVED ON FTA
CARDS
39. ● Whatman FTA products facilitate
collection, transportation,
purification and long-term room
temperature storage of nucleic
acids, all on a single device.
● FTA technology has the ability to
lyse cells, denature protein,
removal of contaminants, and
protects DNA from degradation.
● On FTA Cards small area is
encircled for spotting of
blood/body fluids and this method
is used to obtain DNA from the
following types of samples:
● Peripheral blood Buccal cells
Other body Fluids
50. Principle
The sample is boiled in a solution
containing minute beads of a chemical
called Chelex.
The boiling causes the cells to lyse,
releasing the DNA. The Chelex binds to the
extraneous cellular material, and the entire
“complex” is removed by centrifugation,
leaving the DNA in the supernatant.
Since the high temperatures disrupt the
two strands of the DNA, generating single-
stranded molecules, this extraction process
is generally reserved for polymerase chain
reaction (PCR)-based typing techniques.
51.
52. Procedure Collection of Cells (e.g., buccal cells, liquid blood)
Pipet 10 ml of liquid sample into the polypropylene test tube
For buccal cells, rinse your mouth with 10 ml of 1× PBS solution and vigorously
swish against your cheeks for 10 s. Expel the PBS solution back into the labeled
15 ml polypropylene test tube over the sink.
OR
If sterile swabs are available, place the swab inside your mouth and press it firmly
against the inside of your cheek. Roll the swab back and forth over the inside
surface of your cheek at least 10 times. Repeat on the other cheek. Place the
swab into a labeled 15 ml test tube containing 2 ml of 1× PBS solution. After
gentle swirling the cells will dislodge from the swab in 10–15 min.
56. Principle
❑ Organic extraction is a general method
used for many situations when stained
fabric or clothing is suspected of
containing biological material.
❑ The stain on the material is cut away from
the fabric, soaked in a warm solution
(stain extraction buffer) to release the
cells from the fabric, incubated with
proteinase K, and the DNA isolated using
organic solvents.
❑ The organic extraction method maintains
the integrity of the DNA (i.e., large
segments are maintained) while
“cleaning” the DNA.
61. QIAamp®DNeasy MINI KIT
ISOLATION OF DNA FROM
• LIQUID BLOOD
• DRIED BODY FLUID STAINS
• FRESH AND FROZEN TISSUES
• DNA FROM TRACE EVIDENCE
(CHEWING GUM)
• CIGARETTE BUTTS
Buffers ATL, AL, AE, AW1 and AW2.
66. FACTORS AFFECTING DNA ANALYSIS FROM
BONE
Morphology of bone
(spongy, brittle, non-
compact bones )
Quality of bone
(degraded, damaged,
burnt, charred,
exhumed, fragmented
bone )
Age of bone (fresh, old
or archived)
67. CLEANING AND DECONTAMINATION OF BONE SURFACE:
Cleaning
(remove soft
tissue/debris)
scraping, brushing, rinsing
with water or sonication
10% bleach
WASH
BUFFER (For
1ml wash
Buffer)
1% SDS 100 μl 25mM EDTA
50 μl 1μl Proteinase K
(20mg/ml) 5μl Sterile Milli-
Q Grade water 845 μl
68. SAMPLING METHODS FOR BONE
Cutting
(wearing PPE)
Sonication
(small bones)
Crushing/Grinding (motar
and pestle)
Milling/Processing with
Liquid Nitrogen
(cryogenic/grinds/
pulverize it by impacting a
magnetic based steel
impactor)
71. ISOLATION OF DNA FROM TEETH (ORGANIC
EXTRACTION METHOD)
Larger teeth with no restorations should be chosen over smaller or restored teeth. Thus, non-restored
molars are the tooth of choice for DNA recovery.
Cleaning of Tooth Surface: 10% bleach
72.
73.
74. AUTOMATED DNA EXTRACTION
❑ Automated DNA Extraction system is a Robotic technology to
isolate the DNA from various sources in an easiest way.
❑ We can process a number of samples simultaneously in less
time as compared to manual procedures.
❑ The system comes with different technologies i.e. Silica based
and magnetic beads-based technology.
75. EZ1® DNA INVESTIGATOR
Magnetic-particle technology combines the speed
and efficiency of silica-based DNA purification with
the magnetic particles.
DNA is isolated from lysates through its binding to
the silica surface of the particles in the presence of a
chaotropic salt.
The particles are separated from the lysates using a
magnet.
The DNA is then washed and eluted either in water
or TE buffer.
The user can choose elution volumes of 40 μl (EZ1
Advanced XL only), 50 μl, 100 μl, or 200 μl
76.
77. The Main Features of the
EZ1 Instrument Include:
Purification of high-quality nucleic acids from 1–6
or 1–14 samples per run.
Small footprint to save laboratory space.
Preprogrammed EZ1 Cards containing ready-to-
use protocols for nucleic acid purification.
Prefilled, sealed reagent cartridges for easy, safe,
and fast setup of EZ1 instruments.
Complete automation of nucleic acid purification,
from opening of reagent cartridges to elution of
nucleic acids, with no manual centrifugation steps.
80. AUTOMATE EXPRESS DNA
ISOLATION SYSTEM
The Automate works on the chemistry of reagents. The
reagents used for DNA extraction are:
Prep Filer Express Forensic DNA Extraction Kit (for
soft tissues)
Prep Filer Express BTA Forensic DNA Extraction Kit (for
hard tissues)
The Prep Filer Express Extraction kit is based on the
property of magnetic particles which efficiently binds the
DNA and a multi-component surface chemistry.
During the washing steps the magnetic particles + DNA
complex remains stable, at the same time it removes
inhibitors and allows the efficient release of DNA during
elution to recover a highest amount of pure DNA.
Thirteen (13) samples can be isolated at one time
81.
82. Body Fluids Protocol For processing the various body
fluids such as: Liquid samples (blood, saliva), Blood on
FTA paper or fabric, saliva or semen on any kind of
fabric, Buccal swabs, etc., the materials required for the
lysis
Bone and Tooth Protocol Sampling of Bone and Tooth
Trace Evidence Protocol Substances which are used for
the DNA isolation could be chewing gum, cigarette butts
and adhesive tapes containing saliva or blood sample.
83. Rapid HIT The Applied Biosystems RapidHIT ID System is a fast and simple-to-
use instrument that produces trusted lab-quality forensic DNA profiles in
as little as 90 minutes.
The system integrates sample preparation and capillary
electrophoresis to generate DNA profiles that are aggregated within
Applied Biosystems RapidLINK Software Software v1.3 and its v1.3.
Features of the Applied Biosystems RapidHIT ID System include:
• Sample processing 90 minutes
• 1-minute hands-on time with integrated sample cartridge
• Consumables tracking through radio frequency identification
(RFID)
• Intuitive touchscreen interface
• Facial recognition and barcode camera
• Fingerprint reader
• Up to twelve months of shelf life for both sample (with RapidHIT ID
ACE GlobalFiler Express Sample Cartridge )and primary cartridges
• FBI NDIS-approved rapid DNA system and booking station device*