04/07/18
Design and Applications in
Molecular Biology Research:
PCR and Primer Design
General knowledge of DNA replication
PCR Background
n Invented in 1982 (Cetus Corp)
n Discovery of Taq polymerase in 1985
n Kary Mullis: Nobel Prize 1993
n Widely used method with wide application
n Many variations of commercial kits
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In vitro DNA amplification mimics the in
vivo DNA replication process
n The exponential synthesis of a DNA
fragment is expressed by (2n - 2n)x
n = no. of cycles
x = no. of copies of original template
Polymerase Chain Reaction
n Method for exponential amplification of DNA or
RNA sequences
n Basic requirements
¨ template DNA or RNA
¨2 oligonucleotide primers complementary to different
regions of the template
¨ heat stable DNA polymerase
¨4 nucleotides and appropriate buffer
PCR
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Cycling Program
Step 1: 94o C for 30
sec
Step 2: 94o C for 15
sec
Step 3: 55o C for 30
sec
Step 4: 72o C for
1.5 min
Step 5: Go to step 2
for 35 times
Step 6: 72o C for 10
min
Step 7: 4o C forever
Step 8: END
PCR
Taq DNA Polymerase
n cloned 94 kDa protein
n optimal temperature 75-80 °C
n limited thermostability
n lacks proofreading function
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The Plateau Effect
Departure from the ideal:
n The point in a PCR where
running more cycles does not
result in a net gain of specific
PCR product
n Amplicons not in plateau-
including nonspecific
fragments may continue to
grow exponentially
n Running PCRs into plateau
may result in high background
or smearing
PCR Mix: Buffer and MgCl2
n A typical reaction buffer for PCR would something like:
¨ 10 mM Tris, pH 8.3
¨ 50 mM KCl
¨ 1.5 mM MgCl2
¨ 0.01% gelatin
n The MgCl2 concentration in the final reaction mixture is usually between 0.5 to
5.0 mM, and the optimum concentration is determined empirically (typically
between 1.0 - 1.5 mM).
n Mg2+ ions:
¨ form a soluble complex with dNTP's which is essential for dNTP
incorporation
¨ stimulate polymerase activity
¨ increase the Tm (melting temperature) of primer/template interaction (i.e. it
serves to stabilize the duplex interaction
n Generally,
¨ low Mg2+ leads to low yields (or no yield) and
¨ high Mg2+ leads to accumulation of nonspecific products (mispriming).
n MgCl becomes important when your template DNA is in TE (EDTA can
chealate Mg removing it from the reaction, thus leaving nothing to
catalyze the reaction. Sometimes, just the addition of MgCl2 will
overcome this effect.
Considerations About Primers
Characteristics of primers: Thoughts on primer design:
Specificity Uniqueness
Specific for the intended Length
target sequence (avoid Base Composition
nonspecific hybridization)
Internal Stability
Stability Melting Temperature
Form stable duplex with Annealing Temperature
template under PCR
conditions Internal Structure
Compatibility
Primers used as a pair shall
Primer Pair Matching
work under the same PCR
condition
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Where do primers come from ?
• Chemically synthesized as ssDNA
• Typically 15-25 nucleotides in length
• The nucleotide sequence determined by
- prior knowledge of target DNA sequence
- reverse translation of protein sequence
Primer design elements
n Primer length
n GC%
n Annealing
n 3’ complementary between primers
n G&C runs at the 3’ end
n Palindrome sequences
Good Primer’s Characteristic
n A melting temperature (Tm) in the range of
52°C to 65°C
n Absence of primer dimerization capability
n Absence of significant hairpin formation
(>3 bp)
n Lack of secondary priming sites
n Low specific binding at the 3' end (ie.
lower GC content to avoid mispriming)
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Uniqueness
There shall be one and only one target site in the template DNA where the
primer binds, which means the primer sequence shall be unique in the
template DNA.
There shall be no annealing site in possible contaminant sources, such as
human, rat, mouse, etc. (BLAST search against corresponding genome)
Template DNA
5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’
G AGTTG
TGCTAAGTT CAGTCAACTGCTAC TGCT
A
Primer candidate 1 5’-TGCTAAGTTG-3’ NOT UNIQUE!
Primer candidate 2 5’-CAGTCAACTGCTAC-3’ UNIQUE!
Length
Primer length has effects on uniqueness and melting/annealing
temperature.
Longer the primer = more unique
Longer the primer = higher melting/annealing temperature.
Generally speaking, the length of primer has to be at least 15 bases
to ensure uniqueness. Usually, we pick primers of 17-28 bases
long. This range varies based on if you can find unique primers
with appropriate annealing temperature within this range.
Base Composition
Base composition affects hybridization specificity, melting/annealing
temperature and internal stability.
• Random base composition is preferred. We shall avoid long (A+T) and
(G+C) rich region if possible.
Template DNA
5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’
T
TGCCCGATCATGCT TGCCCGATCATGC
• Usually, average (G+C) content around 50-60% will give us the right
melting/annealing temperature for ordinary PCR reactions, and will give
appropriate hybridization stability. However, melting/annealing temperature
and hybridization stability are affected by other factors, which we’ll discuss
later. Therefore, (G+C) content is allowed to change.
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Melting Temperature
Melting Temperature, Tm – the temperature at which
half the DNA strands are single stranded and half are
double-stranded.. Tm is characteristics of the DNA
composition; Higher G+C content DNA has a higher Tm
due to more H bonds.
Calculation
(Base Composition): Tm = 59.9 + 0.41*(%GC) - 600/
length
Other more accurate methods are available.
Primer Annealing Temperature
Simple Method (Thein and Wallace 1986):
A and T = 2o C
G and C = 4o C
So, add up the number of A and T in the primer and
multiply by 2 and then add up the number of G and
C and multiply by 4. Add these two number together
to get the approximate annealing temperature.
Exercise:
If the following was your primer set (below) using the method above,
what would be the annealing temperature you would use for the reaction:
Forward ATG TGT GAC GAT GAG GTT GC
Reverse TGG CTG GGG CAT TGA AGG TC
Stringency in Primer Annealing
Stringency determines the specificity of the amplified DNA
product. Tanneal is the most significant factor affecting the
stringency in primer annealing.
• Tanneal : too low à less stringent à primer matches elsewhere
too high à more stringent à primer may fail to match
Other factors:
• GC%: GC pairs are more stringent than AT paris
• Salt & Buffer
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Internal Structure
If primers can anneal to themselves, or anneal to each other rather than
anneal to the template, the PCR efficiency will be decreased dramatically.
They shall be avoided.
However, sometimes these 2° structures are harmless when the annealing
temperature does not allow them to take form. For example, some dimers or
hairpins form at 30 °C while during PCR cycle, the lowest temperature only
drops to 60 °C.
Primer Pair Matching
Primers work in pairs – forward primer and reverse primer.
Since they are used in the same PCR reaction, it shall be
ensured that the PCR condition is suitable for both of them.
One critical feature is their annealing temperatures, which
shall be compatible with each other. The maximum
difference allowed is 3 °C. The closer their Tanneal are, the
better.
Summary ~ Primer Design Criteria
1. Uniqueness: ensure correct priming site;
2. Length: 17-28 [Link] range varies;
3. Base composition: average (G+C) content around 50-60%; avoid long
(A+T) and (G+C) rich region if possible;
4. Optimize base pairing: it’s critical that the stability at 5’ end be high
and the stability at 3’ end be relatively low to minimize false
priming.
5. Melting Tm between 55-80 °C are preferred;
6. Assure that primers at a set have annealing Tm within 2 – 3 °C of
each other.
7. Minimize internal secondary structure: hairpins and dimmers shall be
avoided.
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Diagram for PCR Primer Design
Sequence from
which to choose
primers
Results of search,
PCR reaction Primer including suggested
annealing temperatures
parameters
Design shown in list
Primer Selection
Rules
Primer design is an art when done by human beings, and a far
better done by machines.