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| Polymerase Chain Reaction |
DNA Extraction
The standard method of DNA extraction is applied to living beings, plants, crops, etc. Cells are first denatured using detergents
such as CTAB buffer, Tris-Cl, and EDTA. Centrifugation is done to remove the remaining cell debris. Proteins are denatured using proteinase K and then
precipitated by using organic solvents like chloroform: isoamyl alcohol.
Denatured proteins are then again centrifuged and washed to remove the
remaining debris. Then RNA is removed by RNAse treatment. Then further ethanol is used to precipitate
DNA in concentrated form. After centrifugation DNA sample is again dissolved in
TE buffer.
The yield of DNA is measured on gel electrophoresis
which separates the samples based on size. When the sample is passed on
agarose gel, the negatively charged phosphate binds to the positive electrode
when the current passes through the gel. Then, the Spectrophotometer measures the
DNA quantity through light absorbed at various wavelengths. DNA has an absorption
concentration of 260 or 280nm.
Plant DNA Extraction:
The process of DNA extraction from plants, performed in molecular
labs takes some steps which are:
Cell lysis
DNA was extracted by using the CTAB method. There are
different methods of cell lysis. Freeze cells and the thawed cell content. French
Press Method: Hold cell mass and press tightly with force. Sonication: High
sound waves break the cell. The easy and convenient method of cell lysis is
using the CTAB method: Grinding leaves with liquid nitrogen. Preheat the CTAB buffer. Plant leaves are washed
with ethanol and allowed to evaporate. The leaves are ground with CTAB buffer
and transferred into the Eppendorf tube.
Removal of residual components
The sample is then centrifuged and washed with
chloroform: isoamyl alcohol to remove the remaining residues. Then again
centrifugation and isopropanol are added to remove salts and other chemicals.
Again spin the sample. In the end, ethanol is added to precipitate DNA in its pure form. Then centrifugation is
done to get the pellet that contains the original DNA of the plant sample that we
have extracted. TE buffer is added to
preserve DNA for a long time.
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PCR
Polymerase Chain Reaction is a technique that amplifies a specific segment of DNA. The region we want to amplify is known as target DNA. Primers
are 20 base pairs of long nucleotides. The target region is covered by two primers.
The two primers are bound to opposite ends of template DNA and cover target DNA
that needs to be amplified.
Steps of PCR
There are three steps performed by PCR:
Denaturation: Denaturation is an initial step in PCR.
Denaturation is done at a temperature of 90-95C for 3o seconds. In this step, heating is done to break double-stranded DNA into a single strand. The phosphate
hydrogen bonds between base pairs are broken down.
Annealing:
Primers are annealed in the second step. After denaturation, template DNA
is present in the form of a single strand. Primers attach to the single-strand
DNA due to complementary base pairing making it double-stranded. To
enable specific and appropriate primer hybridization, the proper temperature should
be maintained. Annealing should be performed at 70C.
Extension: The
DNA polymerase attaches to the template-primer hybrid and synthesizes the DNA
strand. Taq polymerase, heat-stable DNA polymerase performs the synthesis of DNA
strands. Taq polymerase is a heat-stable enzyme that comes from the bacterium Thermus aquaticus. This process is performed at a temperature of 70-80C. In this step,
the nucleotides are added in 5’-3’ orientation to the opposite strand of
template DNA, after primer attachment.
Types of PCR
Other than standard and qualitative amplification of
target DNA, there are several different types of PCR for amplifying.
Multiplex PCR
The technique is used to analyze several target DNA
by amplifying in a single PCR thermocycler simultaneously.
Nested PCR
This is an improved method. This technique functions
to prevent non-specific product binding which is caused due to amplifying
unusual primer-target DNA binding. This PCR reaction is completed in two
steps. A single pair of primers is used
in the first step to create DNA which serves as a target for the following response from PCR.
Reverse transcriptase PCR
RT-PCR uses mRNA as the precursor material and
reverses transcriptase to transfer mRNA into complementary DNA. Then, using
standard PCR, cDNA is amplified.
Quantitative PCR
This PCR measures the quantity and concentration of
DNA or RNA in the sample.
Hot-Start PCR
It can eliminate the non-specific binding
of target DNA to produce non-specific amplification at a lower temperature.
Touchdown PCR
Throughout the first two phases of amplification, the
annealing temperature is maintained at 3-10C above the expected Tm, and it drops
during the following cycles. There would be greater specificity for primer attachment
during the two cycles of annealing at high temperature, while in the next
cycles, low temperature enables a better-amplified product.
Assembly PCR
It is a process of assembling nucleotides. This PCR
is performed on short segments of DNA with larger overlapping segments and then
combining more segments of DNA. In this way assembly, PCR aids in the synthesis of
long segments of DNA.
Real-time PCR
Real-time PCR involves both qualitative and
quantitative analysis. As the amplification process moves forward, it enables the quantitative estimation of target DNA. This PCR process uses non-specific dyes such as SYBR green that produce fluorescence upon amplification.
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