Nucleic Acid Design Applications

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Nucleic Acid Design Applications Polymerase
Chain Reaction (PCR) Calculating Melting
Temperature (Tm) PCR Primers Design
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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."
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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
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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-
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3CTACGATCTTA-5
5-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-
3 ACTAGTACAGCTACTACGATCT
TA-5
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• PCR uses the following reagents to AMPLIFY
  sections of DNA
• 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.

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Double-Stranded DNA
HEATING
3 5
5 3
PCR Primers
Single-Stranded DNA
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Double-Stranded DNA
5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3 3-CCT
ACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTT
GGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5
HEAT (95ºC, 30 seconds)
Single-Stranded DNA
5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3 3
-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTC
CGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5
COOL (60ºC, 30 seconds)
PCR Primer Annealing
5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3

3-CCCTCCCCCACCGACCCCA-5 5-GGATGGA
ACACTGGGGGGA-3 3-CCTACCTTGTGACCCCCCTCGGCTATGGGTC
CTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCG
ACCCCA-5
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HEAT (72ºC, 30 seconds)
Polymerase Elongation
5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3
CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTT
GGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5 5-GGATGGA
ACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGT
TATCCACCTCAGGGA 3-CCTACCTTGTGACCCCCCTCGGCTATGGGTC
CTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCG
ACCCCA-5
DNA Synthesis after 1 cycle of PCR 1 double
stranded DNA is now 2 copies
5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3 3-CCT
ACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTT
GGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5 5-GGATGGAA
CACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTT
ATCCACCTCAGGGAGGGGGTGGCTGGGGT-3 3-CCTACCTTGTGACC
CCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTG
GAGTCCCTCCCCCACCGACCCCA-5
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• Denaturing Step
• Primer Annealing Step
• Elongation Step

95ºC 30 Sec.
72ºC 30 Sec.
Temperature
60ºC 30 Sec.
Time
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Most PCR applications use 30 cycles (230 1.07
billion), representing an amplification of about
1 billion fold.
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PCR Primers Design 1) Requires knowledge of the
target sequence (i.e. we cant 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)
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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
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Calculating Melting Temperature (Tm)
Tm 4(G C) 2(A T) ºC
5-ACGTGTGTCAGCTGTAGTCG-3 4 x C 7 x G 3 x
A 6 x T
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.
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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 Formamide in the reaction buffer
Amount of Salt (NaCl) in the reaction buffer
Length the DNA hybridization
Percent of GC in the DNA hybridization
This method is commonly used in calculating the
Tm of oligonucleotide probes used in the DNA
microarray assay platform (discussed later).
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• PCR Primers Design
• Designate subsection of gene to amplify.
• OR
• Designate the desired length of the PCR product
  (amplified DNA).

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Example of PCR Primer Design
1) Target Sequence
GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGG
CAACCGTTATCCACCTCAGG
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5-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGG
AGGCAACCGTTATCCACCTCAGG-3
3-CCTCCGTTGGCAATAGGTGGAGT-5
5-ATGGAACACTGGGGGGAGCC-3
3-CCTACCTTGTGACCCCCC
TCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGT
CC-5
Amplified Region, which includes Primer Sequences
Primers (WRITTEN 5 to 3 !!!) Forward Primer
ATGGAACACTGGGGGGAGCC Reverse Primer
TGAGGTGGATAACGGTTGCCTCC
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• Computationally, PCR Primer design involves the
  coordination of
• Selecting 2 different primers that are
• Any distance apart (there are length limits in
  PCR!)
• -OR- A specified distance apart (e.g. PCR
  product will be 300 base-pairs long)
• -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.
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PCR Primer Design Demonstration
gtunknown gene provided by Dr. Kane GGATGGAACACTGGG
GGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACC
TCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGGCTGGGGCCAG
CCTCATGGAGGTGGCTGGGGCCAACCTCATGGAGGTGGCTGGGGTCAGC
TCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGCTGGGGACAGCC
ACATGGTGGTGGAGGCTGGGGTCAAGGTGGTACCCACGGTCAATGGAACA
AACCCAGTAAGCCAAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGC
AGCTGGAGCAGTGGTAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCC
ATGAGCAGGCCTCTTATACATTTTGGCAGTGACTAT
70 Nucleotides Wide
Primer Specifications 60ºC, product bigger that
100 bp, GC clamps.

• Forward Primer GAGCCGATACCCAGGACAGG (20 bases,
  Tm 66)
• Reverse Primer CCATGTGGCTGTCCCCAGCC (20 bases,
  Tm 68)

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• PCR Primer Design Process.
• Establish Specifications
• Target Gene
• PCR Product Size (not needed)
• Tm of Primer pair (must be nearly/exactly the
  same)
• 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).