Molecular genetics plays an important role in medicine by allowing the study of inherited diseases, mapping disease genes, analyzing how genes cause disease, and diagnosing and treating genetic conditions. DNA isolation is the process of extracting DNA from a source, such as whole blood, cultured cells, or tissues, and separating it from other cellular components. Isolation methods involve lysing cells, removing proteins and contaminants, and recovering purified DNA. The choice of isolation method depends on factors like the DNA quantity and purity needed for downstream applications. DNA purification further separates DNA from other molecules and evaluates the extracted DNA.
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
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
A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries will be discussed: genomic libraries and complementary DNA (cDNA) libraries.
Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome.
In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another.
RNA- A polymer of ribonucleotides, is a single stranded structure. There are three major types of RNA- m RNA,t RNA and r RNA. Besides that there are small nuclear,micro RNAs, small interfering and heterogeneous RNAs. Each of them has a specific structure and performs a specific function.
There are 'n' number of DNA isolation methods depending on the sample type, final use of DNA product, etc. This presentation gives an overall idea about different methods of DNA isolation in a simplified way.
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
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
A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries will be discussed: genomic libraries and complementary DNA (cDNA) libraries.
Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome.
In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another.
RNA- A polymer of ribonucleotides, is a single stranded structure. There are three major types of RNA- m RNA,t RNA and r RNA. Besides that there are small nuclear,micro RNAs, small interfering and heterogeneous RNAs. Each of them has a specific structure and performs a specific function.
There are 'n' number of DNA isolation methods depending on the sample type, final use of DNA product, etc. This presentation gives an overall idea about different methods of DNA isolation in a simplified way.
Basics of DNA isolation, What is chemistry behind it. Presently the laboratory of animal science department ,Göttingen university using this technique for dna isolation in pig blood sample.
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Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
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2. Importance of Molecular Genetics
Genetics is playing an important role in the practice of clinical
medicine.
- Medical genetics involves any application of genetics to
medical practice, it thus includes:
Studies of the inheritance of disease in families.
Mapping of disease genes to specific locations on
chromosomes
Analysis of the molecular mechanisms through which genes
cause disease
Diagnosis and treatment of genetic disease (ex. Gene therapy)
3. DNA Isolation
DNA isolation: is an extraction process of DNA
from various sources.
The aim: is to separate DNA present in the nucleus
of the cell from other cellular components.
4. Application of DNA isolation:
It is needed for genetic analysis which used
for:
1- scientific: use DNA in number of Applications , such
as introduction of DNA into cells & animals or plants for
diagnostic purposes (gene clonining)
2- Medicine: is the most common. To identify point sources
for hospital and community-based outbreaks and to predict
virulence of microorganisms
3- forensic science: needs to recover DNA for
identification of individuals ,( for example rapists, petty
thieves, accident , or war victims) , and paternity
determination.
5. Many different methods and technologies are
available for the isolation of genomic DNA.
All methods involve:
A. disruption and lyses of the starting material
followed by
B. Removal of proteins and other contaminants and
finally
C. Recovery of the DNA
6. To choice of a method depends on many
factors:
A. The quantity and molecular weight of the
DNA
B. The purity reqired for application
C. The time and expense
7. Sample Collection
A- Source: Sample can be isolated from any living or dead
organism
Common sources for DNA isolation include:
Whole blood
Buffy coat
Bone material
Buccal cells
Cultured cells
Amniocytes or amniotic fluid
Sputum, urine, CSF, or other body fluids
8. Sample Collection
B. Sample age:
May be fresh or has been stored .
Stored sample can come from:
Archived tissue samples ,
Frozen blood or tissue (biopsy
material) ,
Exhumed bones or tissues &
Ancient human sample.
Dried blood spots
9. DNA Purification && QQuuaannttiiffiiccaattiioonn
Separating DNA from other cellular components such as
proteins, lipids, RNA, etc.
Avoiding fragmentation of the long DNA molecules by
mechanical shearing or the action of endogenous
nucleases
Effectively inactivating endogenous nucleases (DNase
enzymes) and preventing them from digesting the
genomic DNA is a key early step in the purification
process. DNases can usually be inactivated by use of
heat or chelating agents.
10. Key Steps
Lysis of the cells
Removal of contaminants
includes
Proteins
RNA
Other macromolecules
Concentration of purified
DNA
12. 2. Separate DNA From Crude Lysate
DNA must be separated from proteins and cellular debris.
Separation Methods
a) Organic extraction
b) Salting out
13. a) Separation by Organic Extraction
Traditionally, phenol: chloroform is used to extract
DNA.
When phenol is mixed with the cell lysate, two phases
form. DNA partitions to the (upper) aqueous phase,
denatured proteins partition to the (lower) organic phase.
Phenol: Denatures proteins and solubilizes denatured
proteins
14. b) Separation by Salting Out
At high salt concentration, proteins are dehydrated,
lose solubility and precipitate.
Usually sodium chloride, potassium acetate or
ammonium acetate are used.
Precipitated proteins are removed by centrifugation
DNA remains in the supernatant.
15. Separation by Salting Out
Salting out method:
Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Centrifugation will remove the precipitated proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the desired
buffer.
16. Ethanol precipitation:
-Precipitation of DNA: Absolute Ethanol is layered on the top of
concentrated solution of DNA
- Fibers of DNA can be withdrawn with a glass rod
- Washing of DNA
- Desalt DNA: Most salts are soluble in 70% ethanol
17. 2- Use of Commercial DNA purification kits:
The common lysis solutions contain
A. sodium chloride
B. Trimethamine (also known as tris ) , which is a buffer to
retain constant pH
C. Ethylendiaminetetraacetic (EDTA) , which binds metal
ions
D. Sodium dodecyl sulfate (SDS) which is a detergent .
E. An enzyme used in DNA extraction is protienase K
18. 3- Heat denaturation
Achieved by boiling samples.
Heating of a sample to 100 c releases DNA into the solution
but also denatures it by separating the two strand.
Drawbacks: There are remaining inhibitors in the form of
degraded proteins and other organic compound or ions .
19. 4- Magnetic beads with DNA binding
capacity
Magnetic beads are coated with DNA antibodies or
silica to bind to DNA.
Samples are lyses & and then treated with proteinase K.
The lysates are then applied to the beads.
Resin is subsequently washed & DNA is eluted of it at 65c
Magnetic beads are separated from the sample on a
magnetic stand.
20. Summary of DNA extraction :
There are three basic & two optional steps in a DNA
extraction :
1- Cell lysis , to expose the DNA within .
2- removing membrane lipids by adding a
detergents or surfactants .
3- removing proteins by adding a protease .
4- removing RNA by adding an Rnase.
5- precipitating the DNA with alcohol- usually ice
cold ethanol. In these alcohols , DNA strand will
aggregate together, giving a pellet upon centrifugation .
This step also removes alcohol- soluble salt.
21. DNA Extraction & Purification:
Evaluation
DNA concentration can be determined by measuring the
intensity of absorbance with a spectrophotometers &
comparing to a standard curve of known DNA
concentration.
Measuring the intensity of absorbance of the DNA solution
at wavelength 260nm & 280nm is used as a measure of
DNA purity
DNA purity: A260/A280 ratio: 1.7 – 1.9
DNA concentration (μg/ml): A260 X 50
DNA yield:
DNA conc. X Total volume of DNA solution
23. Measurement of DNA purity
Checking for Degradation DNA
Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation. Good quality DNA should migrate as a
high molecular weight band, with little or no evidence
of smearing.
DNA absorbs UV light at 260 &280 nm & aromatic
proteins absorb UV light at 280 nm Apure sample of
DNA has the 260/280 ratio at 1.8 & is relatively free
from protein contamination.
A DNA preparation that is contaminated with protein
will have a 260/280 ratio lower than 1.8