Plasmid DNA extraction
OBJECTIVES
· To
isolate the plasmid pCAMBIA 1304 and pGEM-T easy from Escherichia coli using either the alkaline lysis method or
commercial extraction kit.
· To
spectrophotometrically quantify and estimate the purity of the extracted
plasmid.
INTRODUCTION
Bacterial cells often possess molecules of closed,
circular DNA, otherwise known as ‘plasmids’. They can also be present at much
lower frequencies in certain eukaryotic cell types, such as yeast. They are
non-essential, self-replicating DNA molecules which are important for the
prokaryotic mobile gene pool.
Plasmids can only exist and replicate within a cell,
where it uses host cell machinery. They consist of small circular
double-stranded DNA and have a huge diversity in size i.e. from 2kb-200kb.
Plasmids are vertically distributed to daughter cells
after host cell division. They can also be transferred horizontally between
different strains of prokaryotic cells, during a process called bacterial
conjugation. Their copy number in bacterial cells varies greatly. The plasmid
type determines copy number; high copy number plasmids can be more than 100
copies per cell, whereas others are limited to very few copies per cell.
Plasmids are important for bacterial evolution and
adaptation to the changing environment, as they carry genes which carry
beneficial traits for the bacterial cell.
Different types of plasmids can coexist in one bacterial
cell. For example, strains such as Escherichia coli can have
three different small plasmids in many copies, and one large single copy
plasmid. Copy number depends on how actively transcribed the plasmids origin of
replication is. If it is extremely active, plasmids can undergo several
replication cycles during a single cell cycle.
Individual plasmids carry very few genes, but they can
carry huge selective advantages in certain environments. For example, plasmids
can contain antibiotic resistance genes, posing a risk to public health.
Plasmids carrying resistance genes are known as R plasmids. Increased use of
antibiotics globally has caused bacterial strains carrying R plasmids to
multiply in size; this means resistant pathogens are becoming more common,
making treatment of certain bacterial infections much more difficult. Another
complication is that certain R plasmids can carry up to 10 resistance genes to
different antibiotics, and these plasmids often contain genes which allow
bacterial conjugation to occur.
Other naturally occurring plasmids include those carrying
the sex factor (F), which allows the cell to perform bacterial conjugation,
replication origins and maintenance functions.
Certain types of plasmids can insert part of their DNA
into the host cell genome, by a process known as integration. These plasmids
can exist in two forms, which are extra chromosomal replicons or integrated
plasmids. These plasmids are known as ‘episodes’.
The mitochondria within human cells also contains a
closed circular DNA molecule; this is known as mtDNA. It encodes 37 genes and
16500bp. The endosymbiont theory states that mitochondria were once an
independent prokaryotic species, which formed a symbiotic relationship with a
larger eukaryotic cell. mtDNA can only be inherited from the mother, and
mutations in it lead to a diverse range of diseases due to varying metabolic
demands in organs of the human body.( https://www.news-medical.net/life-sciences/What-are-Plasmids.aspx)
Different extraction methods
result in different yields and purity of DNA. Some of the extraction methods
have been systematically evaluated for specific applications such as soil and
sediment samples, human microbiome, and fecal samples.
First one is organic extraction. In this conventional, widely used method, cells are lysed and cell debris is usually removed by centrifugation. Then, proteins are denatured/digested using a protease, and precipitated with organic solvents such as phenol, or 1:1 mixture of phenol and chloroform. The protein precipitate is removed following separation by centrifugation. Purified DNA is usually recovered by precipitation using ethanol or isopropanol. At some point in the process, RNAs are degraded through incubation with RNase. In the presence of monovalent cations such as Na+, and at -20°C, absolute ethanol efficiently precipitates polymeric nucleic acids and leaves behind short-chain and monomeric nucleic acid components, including the ribonucleotides from RNase treatment in solution. This method uses hazardous organic solvents, is relatively time-consuming, and residual phenol or chloroform may affect downstream applications such as PCR.
Silica-based technologies are widely employed in current kits. DNA adsorbs specifically to silica membranes/beads/particles in the presence of certain salts and at a defined pH. The cellular contaminants are removed by wash steps. DNA is eluted in a low salt buffer or elution buffer. Cha tropic salts are included in the kit buffers to aid in protein denaturation and extraction of DNA. This method can be incorporated in spin columns and microchips, is cost-effective, has a simpler and faster procedure than the organic extraction, and is suitable for automation. Kits based on this method include Purelink Genomic DNA extraction kit from Thermo Fisher and DNeasy Blood and Tissue Kit from QIAGEN.
Magnetic separation is based on DNA reversibly binding to a magnetic solid surface/bead/particles that have been coated with a DNA binding antibody, or a functional group that interacts specifically with DNA. After DNA binding, beads are separated from other contaminating cellular components, washed, and the purified DNA is eluted using ethanol extraction. This method is rapid, simple to perform and can be automated. However, it can be more costly than other methodologies. Examples of commercially available kits include the Agencourt DNAdvance Kit from Beckman Coulter) and Magnetic Beads Genomic DNA Extraction Kit from Gene aid.
DNA extraction by anion exchange chromatography is based on the specific interaction between negatively charged phosphates of the nucleic acid and positively charged surface molecules on the substrate. DNA binds specifically to the substrate in the presence of low salt, contaminants are removed by wash steps using a low or medium salt buffer, and purified DNA is eluted using a high salt buffer. This technology is most commonly employed in plasmid isolation kits such as PureLink® HiPure Plasmid DNA Purification Kits from Thermo Fisher, QIAGEN plasmid mini/midi kits and Genomic-tip, and NucleoBond® PC kits from Macherey Nagel.
Other methods of DNA extraction include salting out, cesium chloride density gradients, and chelex 100 resin. DNA isolation methods are often modified and optimized for different cell types or sample sources. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are often included in protocols for DNA extraction from plant materials, and are discussed in more detail in "DNA extraction from plant tissue and cells".
However,
a very common technique in molecular biology is commonly referred to as
“minipreps”, which usually use an alkaline lysis method. Minipreps are used to
isolate small quantities of DNA from bacterial colonies to screen colonies for
the correct DNA plasmid. Specific protocols for alkaline lysis differ from
laboratory to laboratory, however they are all based on the same principal.
The
first stage is to grow the selected bacterial colonies in a small volume
(3-5ml) of LB broth containing the selection antibiotic. The bacteria are
pelleted and resuspended in a resuspension buffer. This buffer is often a basic
pH Tris buffer, which helps to denature DNA, and EDTA that binds divalent cations destabilizing the
membrane and inhibiting DNases (enzymes that degrade DNA). In addition, RNases
are also added to degrade the released RNA. Next, the bacteria are lysed with
strong alkali (Sodium Hydroxide (NaOH)) and detergent (Sodium Dodecyl Sulfate
(SDS)). The SDS detergent solubilizes the phospholipids and proteins of the
cell membrane resulting in cell lysis and the release of the cells contents.
The high concentration of sodium hydroxide denatures the genomic and plasmid
DNA, as well as cellular proteins. The cellular DNA becomes linearized and the
strands are separated, whereas the plasmid DNA is circular and remains
topologically constrained (the two strands, although denatured remain
together).
Finally,
a neutralization buffer of potassium acetate is added to neutralize the strong
alkaline conditions. The addition of potassium acetate results in a high salt
concentration that leads to the formation of a white precipitate that consists
of SDS, lipids and proteins. In addition, the neutralization of the solution
allows the denaturation of DNA. The large chromosomal DNA is captured in the
precipitate, whereas the small plasmid DNA remains in solution. The precipitate
and chromosomal DNA is removed by centrifugation. Following centrifugation, the
soluble plasmid DNA can be purified from the solution by various techniques.
The most common is to precipitate the DNA with alcohol (ethanol or isopropanol)
or high salt (ammonium acetate, lithium chloride, sodium chloride or sodium
acetate). Another method is to bind the DNA to a solid support, such as glass
fibers or silica. At high salt concentration and neutral or low pH, DNA
molecules have a high binding affinity for these supports, allowing for the
easy capture and subsequent elution of the DNA.
MATERIALS
Fresh
overnight culture of Escherichia coli
Pipette
tips and micropipettes
1.5 mL
micro centrifuge tubes
SET
buffer
Alkaline
Lysis
3M NaOAc
(pH 4.8)
Isopropanol
70%
Ethanol
RNase A
Refrigerated
microcentrifuge
TE
buffer or sterile deionized water
Refrigerated
centrifuge
Vivianites GF-1 plasmid isolation kit
METHODS
1.
The
inoculation loop was sterilized with flame.
2.
Using
inoculation loop E coli was
transferred to the nutrient broth from culture media.
3.
Two
centrifuge tubes were filled with 1.5mL solutions that contain Ecoli. Each
tubes were contain 1.5mL solutions.
4.
Two
tubes were placed inside the centrifuge and two tubes were centrifuged at top
speed for one min.
5.
Tubes
were removed from centrifuge after stop centrifugation
6.
Then
the bacteria pellet was there inside the tube.
7.
Liquids
that contain inside the tubes was removed using pipette and avoid touching
pellets.
8.
250µL
of resumption solution was added to the centrifuge tube.
9.
It
was repeated for 2nd tube.
10.
Resumption
solution was mixed with bacterial pellet.
11.
250µL
of lysis solution was added to each tubes.
12.
It
was mixed gently inverting the tube 6-8 times.
13.
350µL
of Neutralization solution was added to each tubes.
14.
Again
it was mixed inverting 6-8 times.
15.
Two
tubes were kept inside the centrifuge and it was centrifuged at top speed for 5
minutes.
16.
Two
tubes were removed from centrifuge.
17.
Two
column were kept inside the two collection tubes.
18.
Liquid
or supernatant was removed using pipette.
19.
Supernatant
was transferred to the column.
20.
Column
with collection tubes were centrifuged at top speed for 1 minutes.
21.
The
liquids that contain inside the collection tube was removed.
22.
750µL
of wash solution was added to each tubes.
23.
Column
was placed inside the centrifuge and centrifugation at top speed and 1 minutes.
24.
Again
solutions that inside the collection tubes was removed.
25.
Again
two tubes were centrifuged until dry.
26.
Column
was placed in micro centrifuge tube.
27.
50µL
of elution solution was added to each tubes.
28.
It
was allowed one minutes until absorb to column.
29.
Without
closing tubes again it was centrifuged at top speed and one minutes.
30.
The
columns were removed from micro centrifuged tubes and finally purifies DNA.
31.
It
was stored in 4`c or -20`c.
DISCUSSION
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or
other cell components such as proteins by breaking the cells open. Minipreparation of plasmid DNA is
a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the
alkaline lysis method. The
extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process
of molecular cloning to analyze bacterial clones.
However, the procedure start with Ecoli
bacteria with bacterial colony. There are several materials in this method.
They are set buffer or sucrose EDTA and Tris-cl, rnase-A, alkaline lysis
solution, neutralization solution (ice cold NaOAc), ice cold Iso propanol, 70%
ice cold ethanol, genospin column.
Divalent cations (Mg2+, Ca2+) are essential for
DNase activity and the integrity of the bacterial cell wall. EDTA chelates
divalent cations in the solution preventing DNases from damaging the plasmid
and also helps by destabilizing the cell wall. Glucose maintains the osmotic
pressure so the cells don’t burst and RNase A is included to degrade cellular
RNA when the cells are lysed.
The lysis buffer contains sodium hydroxide
(NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to
solubilize the cell membrane. NaOH helps to break down the cell wall, but more
importantly it disrupts the hydrogen bonding between the DNA bases, converting
the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA)
and your plasmid, to single stranded DNA (ssDNA). This process is called
denaturation and is central part of the procedure, which is why it’s called
alkaline lysis. SDS also denatures most of the proteins in the cells, which
helps with the separation of the proteins from the plasmid later in the
process.
Neutralization solution is addition
of potassium acetate returns decreases the alkalinity of the mixture. Under
these conditions the hydrogen bonding between the bases of the single stranded
DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the
selective part. While it is easy for the the small circular plasmid DNA to
re-nature it is impossible to properly anneal those huge gDNA stretches. This
is why it’s important to be gentle during the lysis step because vigorous
mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal
and contaminate your plasmid prep.
While the double-stranded plasmid can dissolve easily in
solution, the single stranded genomic DNA, the SDS and the denatured cellular
proteins stick together through hydrophobic interactions to form a white
precipitate. The precipitate can easily be separated from the plasmid DNA
solution by centrifugation.
Ice cold isopropanol is used to precipitate DNA from the
solution. The salts neutralize the negative charge of the negatively charged
phosphate in DNA and isopropanol or ethanol removes the hydration shell of H2o
molecules around the phosphate.
Once we
recover the DNA or RNA
pellet from the isopropanol,
wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol for ethanol. It is ok to chill the isopropanol-precipitated sample.
DNA extraction is one of the most modern of the biological
sciences. Scientists and doctors use DNA extraction to diagnose many medical
conditions to genetically engineer both plants and animals. DNA extraction can
also be used to gather evidence in a crime investigation.
DNA
extraction can be used to modify plants, by isolating DNA from organisms with
desirable traits, such as resistance to pesticides, and injecting them into the
genome of the plant. When the plant reaches adulthood, its seeds will inherit
the modified genes. DNA extraction, can also be used to alter animals, from
making them glow-in-the-dark to cloning them. A number of pharmaceutical
products, including hormones and vaccines are made using DNA extraction. It is
also used to verify people’s identity, both to determine genetic relatives and
to investigate suspects of crimes in which genetic material was left at the
scene.
DNA extraction is one of the
most modern of the biological sciences. Scientists and doctors use DNA
extraction to diagnose many medical conditions to genetically engineer both
plants and animals. DNA extraction can also be used to gather evidence in a
crime investigation.
DNA extraction can be used
to modify plants, by isolating DNA from organisms with desirable traits, such as
resistance to pesticides, and injecting them into the genome of the plant. When
the plant reaches adulthood, its seeds will inherit the modified genes. DNA
extraction, can also be used to alter animals, from making them
glow-in-the-dark to cloning them. A number of pharmaceutical products,
including hormones and vaccines are made using DNA extraction. It is also used
to verify people’s identity, both to determine genetic relatives and to
investigate suspects of crimes in which genetic material was left at the scene.
DNA extraction is integral
to the process of genetic modification of plants. Many agricultural companies
use genetic extraction to isolate DNA from organisms with desirable traits,
which they then transplant into the plant’s genome.
This is done by taking a
sample of the organism, extracting the DNA, and then cloning it to make
thousands of copies of the single gene that they are interested in. The
scientists then alter the gene to make it ready to work with the rest of the
plant’s DNA, and then insert it into the nucleus of some plant cells. The plant
cells are grown into adult plants, and their offspring seeds all have the
genetic modifications.
An example is number of
lines of seeds manufactured by the Monsanto Corporation that are immune to the
herbicide Roundup. By making the crops (beets, for example) resistant to
Roundup, that particular herbicide can be sprayed on fields to kill weeds, but
not affect the beet crop.
DNA extraction is also the
first step in genetic engineering of animals. Genetic engineering of animals is
a very broad field that ranges from editing a single gene to transplanting
genes from one animal into another. For example, a Taiwanese research lab
transplanted jellyfish genes into pigs, causing them to glow in the dark. On
the most complex end of the spectrum of animal genetic engineering is cloning,
a process by which genetically identical animals can be made.
DNA extraction is used as the initial step in manufacturing a number of pharmaceuticals. Pharmaceuticals made via recombinant genetics include the Hepatitis B vaccine and human growth hormone (hGh). In addition to a number of other hormones created using DNA extraction, one of the most widely used is insulin
Diagnosis of certain medical conditions can often be made from DNA extracted from a patient. Conditions that can be diagnosed by genetic testing include cystic fibrosis, sickle-cell anemia, and fragile x syndrome, Huntington’s disease, hemophilia A, Down’s syndrome and Tay - Sachs disease. In addition to diagnosing existing diseases, geneticists also commonly test whether a person is a carrier of a particular genetic condition but does not have any symptoms of the disease.
A well-known use for genetic
extraction is genetic fingerprinting, a process that matches genetic material
from an individual with other genetic material available. One example is
paternity testing, to determine someone’s biological father. Another common use
for DNA extraction in identity verification is for forensic purposes. Genetic
material from an individual can be compared to genetic material at a crime
scene, such as blood, for example. Genetic verification has worked both to
place a person at the scene of a crime and to exonerate people falsely
convicted of a crime.
·
DNA
extraction from banana.
1. Banana was chopped
2. One teaspoons of salt was added to the half cup of warm water.
3. Banana with salt water was blend.
4. It was removed to cup.
5. Teaspoon of detergent was added to the banana with salt solution.
6. It was stirred for 5 minutes.
7. It was transferred to the bottle.
8. The bottle was kept inside a sock and spin until mix well.
9. The mixture was poured using strainer.
10. Pour solution was got.
11. Alcohol was added to the pour solution.
12. Then it was displayed some collection of DNA as threads.
13. Using toothpick it was extracted.
REFERENCES
https://www.labome.com/method/DNA-Extraction-and-Purification.html
https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation
https://en.bio-protocol.org/e30
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