Nucleic Acid Design Applications
Polymerase Chain Reaction (PCR)
Calculating Melting Temperature (Tm)
PCR Primers Design
When Science named PCR and the polymerase that it employs as its first
"Molecule of the Year" in 1989, the editor, Daniel Koshland Jr., provided a
succinct explanation of PCR. He wrote:
"The starting material for PCR, the 'target sequence,' is a gene or segment of
DNA. In a matter of hours, this target sequence can be amplified a million fold.
The complementary strands of a double-stranded molecule of DNA are
separated by heating. Two small pieces of synthetic DNA, each complementing a
specific sequence at one end of the target sequence, serve as primers. Each
primer binds to its complementary sequence. Polymerases start at each primer
and copy the sequence of that strand. Within a short time, exact replicas of the
target sequence have been produced. In subsequent cycles, double-stranded
molecules of both the original DNA and the copies are separated; primers bind
again to complementary sequences and the polymerase replicates them. At the
end of many cycles, the pool is greatly enriched in the small pieces of DNA that
have the target sequences, and this amplified genetic information is then
available for further analysis."
PCR Concept: Amplification of a relatively short piece of DNA for manipulation or sequencing.
Driving phenomena of PCR: Heating and Cooling
Heating: Double-stranded DNA “comes apart” when heated to near boiling. This is also called
“denaturing” or “melting”.
Cooling: Complementary DNA “comes together” when cooled. This is also called “renaturing”,
“annealing” or “hybridizing”.
Double-Stranded DNA
COOLING
HEATING
Single-Stranded DNA
Molecular Basis of PCR: Polymerase Activity
A Polymerase is an enzyme that synthesizes DNA.
1) DNA can ONLY be synthesized using the complementary strand!
2) Polymerases synthesize DNA in the 5’ 3’ direction!
5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’
3’CTACGATCTTA-5’
5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’
ACTAGTACAGCTACTACGATCTTA-5’
PCR uses the following reagents to AMPLIFY sections of DNA…
1)
2)
3)
4)
DNA template
Polymerase
Free Nucleotides (which are incorporated during DNA synthesis)
PCR Primers
Primers are two short pieces of DNA (each with a unique sequence) that are
complementary to the two different strands of the DNA template.
In line diagrams, the primers are designated as arrows, where the arrows
point in the direction of 3’ DNA synthesis.
Double-Stranded DNA
This section of the DNA template
will be amplified.
HEATING
3’
5’
PCR Primers
5’
3’
Single-Stranded DNA
Double-Stranded DNA
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
HEAT (95ºC, 30 seconds)
Single-Stranded DNA
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
COOL (60ºC, 30 seconds)
PCR Primer Annealing
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGA-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
HEAT (72ºC, 30 seconds)
Polymerase Elongation
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGA
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
DNA Synthesis after 1 “cycle” of PCR = 1 double stranded DNA is now 2 “copies”
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
95ºC
30 Sec.
Temperature
72ºC
30 Sec.
1) Denaturing Step
2) Primer Annealing Step
3) Elongation Step
60ºC
30 Sec.
Time
95ºC
30 Sec.
95ºC
30 Sec.
95ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
95ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
“THERMOCYCLING”
72ºC
30 Sec.
60ºC
30 Sec.
Most PCR applications use 30 cycles (230 = 1.07 billion),
representing an amplification of about 1 billion fold.
PCR Primers Design
1) Requires knowledge of the target sequence (i.e. we can’t use PCR to
amplify an unknown sequence)
2) Use can designate subsection of gene to amplify.
3) Primers must have similar “melting temperatures” (e.g. 60ºC)
Calculating Melting Temperature (Tm)
Based on thermodynamic binding properties of double-stranded DNA.
G-C pairing has higher energy than A-T binding!
There are many methods for calculating Tm, and generally speaking it is
more important to use the same method (consistency) rather than
making exact predictions (accuracy).
Common method;
Every G or C is assigned a value of 4ºC, and every A or T is assigned a
value of 2ºC.
Tm = 4(G + C) + 2(A + T) ºC
Calculating Melting Temperature (Tm)
Tm = 4(G + C) + 2(A + T) ºC
5’-ACGTGTGTCAGCTGTAGTCG-3’
=4 x C
=7 x G
= 11
=3 x A
=6 x T
=9
Tm = 4(11) + 2(9)
Tm = 62ºC
This method of calculating Tm
is limited to short sequences,
and really only useful in PCR
primer design, where the
annealing temperature is
expected to be near 60ºC.
Calculating Melting Temperature (Tm)
For longer DNA-DNA melting temperature calculations, the following
formula is suggested;
Tm = 81.5 + 16.6log(M of NaCl) + 0.41(%GC) – 0.65(%Formamide) – (500/length)
Amount of Salt (NaCl)
in the reaction buffer
Amount of Formamide
in the reaction buffer
Percent of GC in the
DNA hybridization
Length the
DNA hybridization
This method is commonly used in calculating the Tm of oligonucleotide
probes used in the DNA microarray assay platform (discussed later).
PCR Primers Design
Designate subsection of gene to amplify.
-OR –
Designate the desired length of the PCR product (amplified DNA).
Example of PCR Primer Design
1) Target Sequence:
GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG
2) Note that the complementary strand is present (but not shown in sequence file):
GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG
CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC
3) Primers are extracted from sequence:
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG-3’
3’-CCTCCGTTGGCAATAGGTGGAGT-5’
5’-ATGGAACACTGGGGGGAGCC-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC-5’
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG-3’
3’-CCTCCGTTGGCAATAGGTGGAGT-5’
5’-ATGGAACACTGGGGGGAGCC-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC-5’
Amplified Region, which includes Primer Sequences
Primers (WRITTEN 5’ to 3’ !!!)
“Forward” Primer: ATGGAACACTGGGGGGAGCC
“Reverse” Primer: TGAGGTGGATAACGGTTGCCTCC
Computationally, PCR Primer design involves the coordination of;
1) Selecting 2 different primers that are:
a) Any distance apart (there are length limits in PCR!)
b) -OR- A specified distance apart (e.g. “PCR product will be 300 base-pairs long”)
c) -OR- Span a specific subsequence or “motif”
(e.g. “PCR product includes exons #1 and #2”)
2) Selecting 2 different primers that are nearly/exactly the same melting
temperature (Tm). This is usually 60ºC.
3) Many times PCR Primers are designed with a “GC Clamp”. This simply
means that the 3’ end of the primers is a G or a C, which binds more
tightly (than AT) and the Polymerase initiates synthesis more
efficiently.
4) Finally, better results from PCR reactions occur if the two primers are not
complementary to each other, or contain extensive palindrome
sequence.
PCR Primer Design Demonstration:
>unknown gene provided by Dr. Kane
GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG
xx
GAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGGCTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAACC
TCATGGAGGTGGCTGGGGTCAGCTCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGCTGGGGACAG
xx
CCACATGGTGGTGGAGGCTGGGGTCAAGGTGGTACCCACGGTCAATGGAACAAACCCAGTAAGCCAAAAA
CCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGGTAGGGGGCCTTGGTGGCTACATGCT
GGGAAGTGCCATGAGCAGGCCTCTTATACATTTTGGCAGTGACTAT
70 Nucleotides Wide
Primer Specifications: 60ºC, product bigger that 100 bp, GC clamps.
1) Forward Primer: GAGCCGATACCCAGGACAGG (20 bases, Tm = 66)
2) Reverse Primer: CCATGTGGCTGTCCCCAGCC (20 bases, Tm = 68)
xx
1) Forward Primer: GAGCCGATACCCAGGACAGG
(18 bases, Tm = 60)
2) Reverse Primer: CCATGTGGCTGTCCCCAGCC
(18 bases, Tm = 60)
xx
PCR Primer Design Process.
1)
Establish Specifications
a) Target Gene
b) PCR Product Size (not needed)
c) Tm of Primer pair (must be nearly/exactly the same)
d) GC Clamp (not needed)
2) Identify Candidate Primers
START WITH 20 BASE PRIMER LENGTH!!!
a) “Forward” Primer is derived from Target Sequence (5’-3’)
b) “Reverse” Primer is;
i) To the “right” or “3’ direction” of the Forward Primer
ii) It is Complementary to the Target Sequence (3’-5’)!!!
iii) It must be written 5’-3’ (inverted) to be correct convention.
3) Calculate Tm for each Primer
a) If calculated Tm is NOT at specified temperature, ADD or DELETE bases from end.
* OR *
b) If calculated Tm is NOT at specified temperature, MOVE Primer sequence 1 or more bases.
4) Check Primers for “Primer-pair binding” (i.e. if the are primers significantly complementary with
each other, they will bind to each other and decrease PCR reaction performance).