PCR (Polymerase Chain Reaction)
Polymerase Chain Reaction
is widely held as one of the most important
inventions of the 20th century in molecular
biology. Small amounts of the genetic
material can now be amplified to be able
to a identify, manipulate DNA, detect
infectious organisms, including the viruses
that cause AIDS, hepatitis, tuberculosis,
detect genetic variations, including mutations,
in human genes and numerous other tasks.
PCR involves the following three steps: Denaturation, Annealing and Extension. First,
the genetic material is denatured, converting
the double stranded DNA molecules to
single strands. The primers are then
annealed to the complementary regions
of the single stranded molecules. In
the third step, they are extended by
the action of the DNA polymerase. All
these steps are temperature sensitive
and the common choice of temperatures
is 94oC, 60oC
and 70oC respectively. Good
primer design is essential for successful
reactions.
The important design considerations
described below are a key to specific
amplification with high yield. The preferred
values indicated are built into all
our products by default.
1. Primer Length: It is generally accepted that the optimal
length of PCR primers is 18-22 bp. This
length is long enough for adequate specificity
and short enough for primers to bind
easily to the template at the annealing
temperature.
2. Primer Melting Temperature: Primer Melting Temperature (Tm)
by definition is the temperature at
which one half of the DNA duplex will
dissociate to become single stranded
and indicates the duplex stability.
Primers with melting temperatures in
the range of 52-58 oC generally
produce the best results. Primers with
melting temperatures above 65oC
have a tendency for secondary annealing.
The GC content of the sequence gives
a fair indication of the primer Tm.
All our products calculate it using
the nearest neighbor thermodynamic theory,
accepted as a much superior method for
estimating it, which is considered the most
recent and best available.
Formula for
primer Tm calculation:
Melting Temperature Tm(K)={ΔH/
ΔS + R ln(C)}, Or Melting Temperature
Tm(oC) = {ΔH/
ΔS + R ln(C)} - 273.15 where
ΔH
(kcal/mole) : H is the Enthalpy.
Enthalpy is the amount of heat energy
possessed by substances. ΔH is
the change in Enthalpy. In the above
formula the ΔH is obtained by
adding up all the di-nucleotide pairs
enthalpy values of each nearest neighbor
base pair.
ΔS
(kcal/mole) : S is the amount of
disorder a system exhibits is called
entropy. ΔS is change in Entropy.
Here it is obtained by adding up all
the di-nucleotide pairs entropy values
of each nearest neighbor base pair. An additional salt correction
is added as the Nearest Neighbor parameters
were obtained from DNA melting studies
conducted in 1M Na+ buffer and this
is the default condition used for all
calculations.
ΔS
(salt correction) = ΔS (1M NaCl
)+ 0.368 x N x ln([Na+])
Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.
[Na+] calculation:
[Na+] =
Monovalent ion concentration +4 x free
Mg2+.
3. Primer Annealing Temperature: The primer melting temperature
is the estimate of the DNA-DNA hybrid
stability and critical in determining
the annealing temperature. Too high
Ta will produce insufficient
primer-template hybridization resulting
in low PCR product yield. Too low Ta may possibly lead to non-specific products
caused by a high number of base pair
mismatches,. Mismatch tolerance is found
to have the strongest influence on PCR
specificity.
Ta = 0.3 x Tm(primer) + 0.7
Tm (product) – 14.9
where,
Tm(primer) = Melting Temperature of the primers
Tm(primer) = Melting Temperature of the primers
Tm(product)
= Melting temperature of the product
4. GC Content: The
GC content (the number of G's and C's
in the primer as a percentage of the
total bases) of primer should be 40-60%.
5. GC Clamp: The
presence of G or C bases within the
last five bases from the 3' end of primers
(GC clamp) helps promote specific binding
at the 3' end due to the stronger bonding
of G and C bases. More than 3 G's or
C's should be avoided in the last 5
bases at the 3' end of the primer.
6. Primer Secondary Structures: Presence of the primer secondary structures
produced by intermolecular or intramolecular
interactions can lead to poor or no
yield of the product. They adversely
affect primer template annealing and
thus the amplification. They greatly
reduce the availability of primers to
the reaction.
i)
Hairpins: It is formed by
intramolecular interaction within the
primer and should be avoided. Optimally
a 3' end hairpin with a ΔG of
-2 kcal/mol and an internal hairpin
with a ΔG of -3 kcal/mol is tolerated
generally.
ΔG
definition: The Gibbs Free Energy
G is the measure of the amount of work
that can be extracted from a process
operating at a constant pressure. It
is the measure of the spontaneity of
the reaction. The stability of hairpin
is commonly represented by its ΔG
value, the energy required to break
the secondary structure. Larger negative
value for ΔG indicates stable,
undesirable hairpins. Presence of hairpins
at the 3' end most adversely affects
the reaction.
ΔG
= ΔH – TΔS
ii) Self Dimer:
A primer self-dimer is formed by intermolecular interactions
between the two (same sense) primers,
where the primer is homologous to itself.
Generally a large amount of primers are
used in PCR compared to the amount of
target gene. When primers form intermolecular
dimers much more readily than hybridizing
to target DNA, they reduce the product
yield. Optimally a 3' end self dimer with
a ΔG of -5 kcal/mol and an internal
self dimer with a ΔG of -6 kcal/mol
is tolerated generally.
iii) Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
iii) Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
7. Repeats: A repeat
is a di-nucleotide occurring many times
consecutively and should be avoided
because they can misprime. For example:
ATATATAT. A maximum number of di-nucleotide
repeats acceptable in an oligo is 4 di-nucleotides.
8. Runs: Primers
with long runs of a single base should
generally be avoided as they can misprime.
For example, AGCGGGGGATGGGG has runs
of base 'G' of value 5 and 4. A maximum
number of runs accepted is 4bp.
9. 3' End Stability: It is the maximum ΔG value of
the five bases from the 3' end. An unstable
3' end (less negative ΔG) will
result in less false priming.
10. Avoid Template Secondary
Structure: A
single stranded Nucleic
acid sequences is highly unstable and
fold into conformations (secondary structures).
The stability of these template secondary
structures depends largely on their
free energy and melting temperatures(Tm).
Consideration of template secondary
structures is important in designing
primers, especially in qPCR. If primers
are designed on a secondary structures
which is stable even above the annealing
temperatures, the primers are unable
to bind to the template and the yield
of PCR product is significantly affected.
Hence, it is important to design primers
in the regions of the templates that
do not form stable secondary structures
during the PCR reaction. Our products
determine the secondary structures of
the template and design primers avoiding them.
11. Avoid Cross Homology:
To
improve specificity of the primers it
is necessary to avoid regions of homology.
Primers designed for
a sequence must not amplify other genes
in the mixture. Commonly, primers are
designed and then BLASTed to test the
specificity. Our products offer a better
alternative. You can avoid regions of
cross homology while designing primers.
You can BLAST the templates against
the appropriate non-redundant database
and the software will interpret the
results. It will identify regions
significant
cross homologies in each template and
avoid them during primer search.
Parameters
for Primer Pair Design
1. Amplicon Length: The amplicon length is dictated by the
experimental goals. For qPCR, the target
length is closer to 100 bp and for standard
PCR, it is near 500 bp. If you know
the positions of each primer with respect
to the template, the product is calculated
as: Product length = (Position of antisense
primer-Position of sense primer) + 1.
2. Product Position: Primer can be located near the 5' end,
the 3' end or any where within specified
length. Generally, the sequence close
to the 3' end is known with greater
confidence and hence preferred most
frequently.
3. Tm of Product: Melting Temperature (Tm)
is the temperature at which one half
of the DNA duplex will dissociate and
become single stranded. The stability
of the primer-template DNA duplex can
be measured by the melting temperature
(Tm).
4. Optimum Annealing Temperature
(Ta Opt): The formula
of Rychlik is most respected. Our products
use this formula to calculate it and
thousands of our customers have reported
good results using it for the annealing
step of the PCR cycle. It usually results
in good PCR product yield with minimum
false product production.
Ta Opt = 0.3 x(Tm of primer)
+ 0.7 x(Tm of product) -
14.9
where
Tm of primer is the melting temperature of the less stable primer-template pair
Tm of product is the melting temperature of the PCR product.
Tm of primer is the melting temperature of the less stable primer-template pair
Tm of product is the melting temperature of the PCR product.
5. Primer Pair Tm Mismatch
Calculation: The two primers
of a primer pair should have closely
matched melting temperatures for maximizing
PCR product yield. The difference of
5oC or more can lead no amplification.
Primer Design using Software
A number of primer design tools are
available that can assist in PCR primer design for new and experienced
users alike. These tools may reduce the cost and time involved in
experimentation by lowering the chances of failed experimentation.
Primer Premier
follows all the guidelines specified for PCR primer design. Primer
Premier can be used to design primers for single templates, alignments,
degenerate primer design, restriction enzyme analysis. contig analysis
and design of sequencing primers.
The guidelines for qPCR primer design vary slightly. Software such as AlleleID and Beacon Designer
can design primers and oligonucleotide probes for complex detection
assays such as multiplex assays, cross species primer design, species
specific primer design and primer design to reduce the cost of
experimentation.
PrimerPlex is a software that can design primers for Multiplex PCR and multiplex SNP genotyping assays.