PCR

PCR reaction mixture must include:

1. DNA template
2. two PCR primers
3. DNA polymerase
4. Deoxynucleoside triphosphates (dNTPs)
5. buffer solution

DNA template in PCR amplification

DNA from a variety of sources may be used as the supplier of the DNA template for 3
basic steps of the polymerase chain reaction. PCR is a highly sensitive technique
and requires only one or two DNA templates for successful amplification.
Nevertheless, the optimal quantity of DNA for PCR amplification depends on the
composition of the DNA and utilized DNA polymerase. It is important to optimize
DNA input, because high amounts lead to nonspecific amplification whereas low
amounts reduce the production of amplicons.
Usually, 0.1–1 ng of plasmid DNA or 5–50 ng of genomic DNA is sufficient for a 50
µL PCR reaction mixture.

3 basic steps of PCR process:

The polymerase chain reaction is a three step cycling process consisting of defined sets of
times and temperatures.

3 basic PCR steps include:

1. denaturation step;
2. annealing step;
3. extension (elongation) step.

Each of these polymerase chain reaction steps is repeated 30–40 times (cycles).

Function of dNTPs in PCR

Deoxynucleoside triphosphates (dNTPs) are the building blocks from which the DNA
polymerase synthesizes a new DNA strand during successive cycles of PCR amplification.
dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP.

For optimum base inclusion, dNTPs are usually added to the PCR mixture in equimolar
amounts.

PCR buffer composition

A buffer of the PCR reaction mixture serves as a chemical environment to maintain an
activity and stability of the DNA polymerase. The buffer pH is usually between 8.0 and 9.5
and is often stabilized by Tris-HСl.

A common component of the Taq polymerase buffer is potassium ion from KCl, which
promotes primer annealing.

The buffer concentration of magnesium ions (Mg2+ from MgCl2) is another crucial factor
for the proper functioning of the DNA polymerase. Mg2+ ions serve as a cofactor for the
enzyme.

In addition, magnesium ions facilitate formation of the complex between the primers and
DNA templates by stabilizing negative charges on phosphate backbones.

Generally, a magnesium concentration is 1.5 mM.

A higher magnesium concentration is linked with a higher output, but a lower specificity,
while a lower magnesium concentration gives decreased enzyme activity and increased
specificity.

Role of DNA primers in PCR

The PCR requires the knowledge of DNA sequences that flank the DNA template.

Primers are short nucleotide sequences (approximately 15–30 bases) that base pair to a
specific portion of the DNA being replicated.

In order for hybridization to occur, the primer nucleotides must have a sequence that is
complementary to the 3′ end of each strand of the DNA target sequence, and the 3′ ends of
the hybridized primers should point toward one another.

The sequences of the primers are very important for the polymerase chain reaction because
the reaction cycle has the specific temperatures used in the heating and cooling stages.
Besides, a great excess of the primers in the PCR reaction mixture cause them more likely to
encounter a partially complementary primer than a perfectly complementary DNA template.
So, primer complementarity has to be avoided.

Two oligonucleotide primers have to be designed and chemically synthesized.

Additional PCR reaction components

To improve PCR results, there are various PCR enhancers. These components of polymerase
chain reaction can relieve secondary DNA structure, lower temperature of template
denaturation or stabilize DNA polymerases.

Commonly used PCR additives include dimethyl sulfoxide (DMSO), ammonium sulfate,
polyethylene glycol, bovine serum albumin (BSA), gelatin, N,N,N-trimethylglycine, and
glycerol.

Imperfections of Taq polymerase:

1. Taq polymerase lacks proofreading activity and unable to correct wrong incorporated
nucleotide bases. On average, these errors occur approximately once per 9000
nucleotides. This might be important for some applications, such as cloning, because
the product of the polymerase chain reaction may not be a completely precise copy of
the original sequence.

2. Another imperfection of Taq polymerase is that it can only efficiently amplify

fragments of a few thousand base pairs. The DNA fragment to be amplified should
not be greater than about 3 kb in length. Usually, they are less than 1 kb.
Amplification of very long fragments (up to 40 kb) requires special methods.

These problems can be solved by using other thermostable polymerases, such

as Pfu and Pwo DNA polymerases. These enzymes have proofreading activity.

PCR Primers Requirements:

The slide you've shared provides important guidelines for designing PCR primers, which are
short DNA sequences used in the PCR (Polymerase Chain Reaction) process to amplify
specific parts of DNA. Here's a simplified explanation of each point:
1. Polynucleotide stretches should be avoided: Avoid having sequences in the primer
that repeat the same nucleotide, especially guanine (G), three or more times
consecutively. This repetition can cause issues due to the nucleotides sticking
together, which might interfere with the PCR.
2. Avoid secondary structures: Make sure that the primers don’t have internal
structures like hairpins (loops formed when a single DNA strand pairs with itself).
These structures can prevent the primer from binding properly to the target DNA.
3. Final concentration of each primer: The amount of primer in the PCR mix should
generally be between 10 and 500 nanomolar (nM), with an optimal range of 100-300
nM. This concentration helps ensure efficient and specific DNA amplification.
4. Higher primer concentrations and secondary priming: Using too much primer can
lead to non-specific binding, meaning the primers might start binding to and
amplifying parts of the DNA that you’re not interested in, leading to unwanted PCR
products.
5. Primer length for large DNA products: If you’re trying to amplify a long DNA
segment (over 5 kilobases), use primers that are at least 25 nucleotides long. Also, the
temperature at which the DNA strands melt apart (Tm) should be higher than 68°C to
ensure specificity.
6. Engineering restriction sites: If you need to add restriction sites (specific DNA
sequences where enzymes cut the DNA) to the ends of your PCR product, add an
extra six nucleotides beyond the restriction site. This addition helps the enzyme
recognize and cut the DNA more efficiently.
These guidelines help in designing effective primers that increase the accuracy and efficiency
of PCR, a crucial technique used in genetics, biology research, and medical diagnostics.
The slide u shared provides specifications for designing primers for PCR (Polymerase
Chain Reaction). Here's a simple explanation of each point:
1. Length of 18-24 bases: Primers should be between 18 and 24 nucleotides long. This
length is optimal for ensuring that the primer binds specifically to the intended target
in the genome, which typically won't occur more than once, minimizing off-target
effects.
2. 40-60% G/C content: The guanine (G) and cytosine (C) bases in the primer should
make up 40-60% of its total base content. G and C pair more strongly (three hydrogen
bonds) than adenine (A) and thymine (T) (two hydrogen bonds), which helps the
primer to bind more stably to the DNA template.
3. Start and end with 1-2 G/C pairs: Primers should start and end with one or two G/C
pairs. This enhances the stability of the primer binding at the ends, which is crucial
for efficient amplification.
4. Melting temperature (Tm) of 50-60°C: The melting temperature is the temperature
at which half of the DNA duplex dissociates to single strands. A Tm of 50-60°C is
ideal for most PCR reactions, providing a balance between specificity and efficient
binding.
5. Primer pairs should have a Tm within 4°C of each other: To ensure that both the
forward and reverse primers bind to the template at similar conditions, their melting
 temperatures should be closely matched, within 4°C. This uniformity helps in efficient
and specific amplification.
6. Primer pairs should not have complementary regions: Primers should not have
sequences that can bind to each other because this can lead to the formation of primer
dimers, where primers bind to each other instead of the template. This uses up primers
unproductively and reduces the yield of the desired product.
7. Avoid primer dimers: This reiterates the importance of designing primers that do not
bind to each other. Primer dimers can significantly interfere with the PCR by reducing
the amplification of the target sequence.

The slide you've shared outlines the typical stages involved in conducting a Polymerase
Chain Reaction (PCR) procedure. Each stage is crucial for the success of the PCR,
which is used to amplify specific regions of DNA or RNA. Here’s a simple explanation of
each stage:

Identify the target gene: Before beginning PCR, you need to determine which gene or
segment of DNA you want to amplify. This could be based on research goals, such as
studying a particular gene associated with a disease.

Design primers: Once the target gene is identified, you need to design short DNA sequences
called primers that specifically bind to the start and end of the region you want to amplify.
The design of these primers is crucial for the specificity and efficiency of the PCR.
Select tissues: Choose the tissue sample from which DNA or RNA will be extracted. The
choice of tissue depends on where your gene of interest is expressed or what is most relevant
to your study.
Extract Nucleic Acid (DNA or RNA): Extract the genetic material (either DNA or RNA)
from the tissue samples. This involves breaking open the cells and purifying the DNA or
RNA from other cellular components.
Synthesize cDNA (if RNA is the target): If RNA is the target, convert it into complementary
DNA (cDNA) using an enzyme called reverse transcriptase. This is necessary because PCR
can only amplify DNA.
Amplify the target using specific primers: Use the PCR machine to amplify the target
DNA. This involves cycles of heating and cooling that denature the DNA, anneal the primers,
and extend the new DNA strands, effectively doubling the amount of DNA with each cycle.
Visualize products using gel electrophoresis (Endpoint PCR): After PCR, the amplified
DNA products are separated using gel electrophoresis, which allows you to see the size of the
fragments that have been amplified. This helps verify that the correct target has been
amplified.
These stages form the core of the PCR process, enabling researchers to generate large
quantities of a specific DNA or RNA segment from a small initial sample, facilitating further
genetic analysis and experimentation.

The slide you've shared outlines the thermocycling conditions for a typical PCR (Polymerase
Chain Reaction) process, along with a diagram showing the temperature cycles over time.
Here's a breakdown of each stage and what it represents in the PCR process:
1. Initial Denaturation at 94°C for 4 minutes: This initial step is used to denature
(separate) the double-stranded DNA into single strands so that the primers can anneal
 to their complementary sequences. The high temperature of 94°C ensures that all the
DNA is fully denatured.
2. Cycling Conditions:
 Denaturation at 94°C for 1 minute: Each cycle begins with this step to
ensure the DNA template strands are separated and ready for primer annealing.
 Annealing at 50-60°C for 30-60 seconds: The temperature is lowered to
allow the primers to bind to their complementary sequences on the single-
stranded DNA. The exact temperature depends on the melting temperature
(Tm) of the primers used.
 Extension at 72°C for 1 minute: At this temperature, the DNA polymerase
enzyme extends the primers, synthesizing new strands of DNA starting from
the primers. This builds new strands complementary to the original template.
3. Final Extension at 72°C for 5 minutes: After completing the cycles, a final
extension step is performed to ensure that any remaining single-stranded DNA is fully
extended.
4. Number of Cycles (35-40 cycles): The process of denaturation, annealing, and
extension is repeated typically 35-40 times to amplify the DNA to a detectable level.
Each cycle theoretically doubles the amount of DNA, leading to exponential
amplification of the target sequence.
5. Hot Start PCR: This term indicates a technique used to improve the specificity and
yield of the PCR. In hot start PCR, a crucial component of the PCR reaction (often the
polymerase enzyme) is inactive at lower temperatures and only activates at higher
temperatures (e.g., after the initial denaturation step). This prevents non-specific
amplification that might occur at lower temperatures before the thermal cycling
begins.
Temperature has an important effect on the hybridization of the primers to the
template DNA
Other factors are
Salt concentration and Size of template

The slide presents a comparison of two DNA staining methods used in gel electrophoresis:
Ethidium Bromide (EtBr) and SmartGlow PS, using a 250-25Kbp DNA ladder. Ethidium
Bromide, a traditional DNA stain that fluoresces orange under UV light, is known for its
mutagenic properties, necessitating careful handling. SmartGlow PS, shown on the right side
of the slide and glowing green, serves as a safer alternative, offering comparable visualization
of DNA bands with reduced health risks. This comparison highlights the effectiveness of both
stains in visualizing PCR products, while underscoring the safety benefits of newer staining
technologies like SmartGlow PS.

The slide depicts the setup and process of gel electrophoresis, a method used to separate DNA
fragments based on their size. The diagram shows an agarose gel placed on a UV-transparent
plastic support, with wells at one end where DNA samples are loaded. After running the
electrophoresis, the gel is soaked in a solution containing Ethidium Bromide (EtBr), a DNA
stain, for 15 minutes. This allows the EtBr to bind to the DNA. When the gel is exposed to
UV light, the DNA bands fluoresce, making them visible. This process helps in visualizing
and analyzing the sizes of the DNA fragments present in the samples, crucial for various
genetic studies and diagnostics.
1. Why supercoiled molecules move faster?
 Supercoiled DNA molecules move faster during gel electrophoresis because
they are more compact than linear or relaxed circular DNA. This compact
structure allows them to navigate through the gel matrix more efficiently.
2. Does Ethidium bromide slow down DNA movement?
 Ethidium bromide can slightly slow down the movement of DNA through a
gel because it increases the mass and size of the DNA by binding to it.
However, this effect is generally minimal and does not significantly affect the
results of gel electrophoresis.
3. How do molecules with secondary structures migrate?
 Molecules with secondary structures such as loops or hairpins may migrate
differently compared to their linear counterparts due to their altered shape and
size. These structures can cause the molecule to migrate slower than expected
based on their length alone.
4. How to make DNA and RNA linear for Agarose Gel electrophoresis?
  To linearize DNA, you can use restriction enzymes to cut circular DNA at
specific sites. For RNA, which is typically already linear, ensure it is fully
denatured before loading onto a gel by treating it with heat or chemicals that
disrupt secondary structures.
5. What if I want to load more samples (volume) on agarose gel?
 To load more volume, you can use wider wells or a thicker gel. However, be
cautious as overloading can lead to poor resolution. Alternatively, concentrate
your samples or use a stacking gel to load more volume without compromising
resolution.
6. How many copies of a template would be in tube after 30 cycles of PCR with a
single primer (Asymmetrical PCR)?
 In asymmetrical PCR, one primer is in excess over the other, leading to the
production of single-stranded DNA. After 30 cycles, the amplification can
theoretically result in over a billion copies of the strand that is primed by the
primer in excess. The exact number can vary based on efficiency and initial
template quantity.
7. Troubleshooting Longer PCRs i.e. ≥4 kb:
 For longer PCR products, ensure using a high-fidelity polymerase suited for
long amplifications, adjust magnesium ion concentration appropriately, and
optimize annealing temperatures. Also, increase extension times
proportionally to the length of the target to allow complete DNA synthesis.
8. Amplifying AT-rich regions or GC-rich regions:
 AT-rich regions may require lower annealing temperatures to avoid premature
dissociation of primers. For GC-rich regions, consider using additives like
DMSO or betaine to help reduce secondary structure formation and improve
primer binding. Adjusting the annealing temperature may also be necessary.
Ammonium sulfate [(NH4)2SO4] is a salt that can enhance the specificity and
yield of PCR by destabilizing weak hydrogen bonds between mismatched primer-
template base pairs. This reduces the formation of nonspecific products and
primer dimers. ➢Ammonium sulfate can also increase the amplification rate of
DNA polymerase and enable more complex PCR systems such as multiplex PCR.
KCl affects the annealing temperature and efficiency of the PCR by influencing
the stability of DNA duplexes. Higher concentrations of KCl can lower the
melting temperature (TM) of DNA and increase the probability of primer
annealing. ➢ However, too high concentrations of KCl can also reduce the
specificity and fidelity of PCR by increasing the chance of mispriming and
mismatch extension. ➢ Itis responsible for the elongation of the primer-DNA
 complex ➢ It works best with shorter DNA fragments but can fail when it comes
to longer DNA fragments.

MgCl2 Increases amplification. ➢ Prevents denaturation of template DNA by increasing

the melting point of DNA. ➢ By directly binding to the phosphate backbone of the DNA, it
prevents denaturation and increases the time for the primer to bind to the target. >Mg
means > Amplification and <specificity

Triton: Triton is a nonionic detergent that reduces PCR inhibition by interfering with
substances such as proteins, lipids, polysaccharides, and metal ions. Triton also prevents the
aggregation of DNA polymerase molecules and enhances enzyme activity and stability. Triton
also stabilizes Mg2+, which is an essential cofactor for DNA polymerase.
DMSO: Dimethyl Sulfoxide can be a principal part of your PCR buffer if the template DNA
segment is abundant in GC (GC rich). It isn’t easy to amplify a DNA template with high GC
content due to its higher chances of forming the secondary hairpin structure. It prevents
hairpin structure formation and improves the sensitivity of the primer for binding.
Amplification Refractory Mutation System:
“A method used to detect a single base change or SNP using sequence-specific primers is
referred to as ARMS or Allele specific PCR.”

1. What if the nonspecific band is of a large size?

 If you see a nonspecific band that is larger than expected, it could be due to
primer-dimer formations or mispriming that occurs at sites other than the
target sequence. To resolve this, you may consider increasing the annealing
temperature to enhance specificity, redesigning the primers, or optimizing the
primer concentration.
2. What if the nonspecific band is of a small size?
 Smaller nonspecific bands could also be due to primer-dimers or other PCR
artifacts. These can be minimized by adjusting PCR conditions such as
reducing the primer concentration, adjusting the magnesium ion concentration
(as Mg^2+ is crucial for primer binding and polymerase activity), or using a
hot-start PCR method where the polymerase is activated at a higher
temperature to reduce non-specific amplification.
RNase H activity:
RNase H activity degrades RNA from RNA-DNA duplexes to enable efficient synthesis of
double-stranded DNA. However, with long mRNA templates, RNA may be degraded
prematurely which can result in truncated cDNA. 27-30/5/2024 Hence, it is generally
beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA
cloning. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored
in qPCR applications because they can enhance the melting of RNA-DNA duplex during the
first cycles of PCR .