DIY Primer Design
Oligonucleotides for Special Applications in
Molecular Biology
Alberto Catalano
Kanematsu Labs, Institute of Haematology
RPAH
alberto.catalano@email.cs.nsw.gov.au
Ph: 9515 7453
http://users.bigpond.net.au/albert/primers.htm
Outline
 DNA Refresher: “DNA 101”
 PCR
 Introduction
 Oligonucleotide Primers for PCR
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Properties
Specificity
Self complementarity & primer-primer interactions
General rules
 Primer Design
 Computer programmes: on internet & on PC
 Special PCR Applications
DNA 101
The “Basics”
B-DNA
2nm
• Common form of DNA
• Formed under high humidity
conditions
• Right-handed double helix
• Major groove & minor groove
• Sugar-phosphate backbones
3.4nm
10
nucleotides
per turn
Ramaswamy H. Sarma 1996
Sugar + Phosphate + Base
Sugar + Phosphate
form the backbone
Base-pairing & DNA Stability
 4 nucleotide bases in DNA
 Cytosine (C) pairs with Guanine (G)
 3 hydrogen bonds
 Strong-pairing
 Adenine (A) pairs with Thymine ( T )
 2 hydrogen bonds
 Weak-pairing
 Stacking forces
 Van der Waals forces
 Influenced by nearest neighbour sequence
Base-pairing
Purines
Pyrimidines
Oligonucleotides
Short single-stranded DNA
Uses for oligonucleotides
 PCR, etc.
 Primer pairs
 Primer sets in multiplex assays
 Probes
 Sequence identification
 Gel shift assays
 Gene technology
 Synthetic genes
 Site-directed mutagenesis
Oligonucleotide Choice
 Sequence
 Specificity
 GC content
 Target sequence location
 Avoiding repeat sequences
 Melting temperature
 Avoid Secondary structures
Specificity
 Approximation of complexity (for a random
sequence)
 1 base = 41 ; 2 bases = 42 ; 3 bases = 43 ; ...
n bases = 4n
 Biological sequences are not random!
 Check the oligos with BLAST
 Need to avoid complementarity with
repetitive sequences in specific organism
 e.g. human Alu sequences, simple repeats
Unwanted Self & Primer-Primer
Interactions
 Primer self-complementarity
 At 3’-end can result in primer-dimer formation
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 Internal homology : stem & loop structures
 Forward & reverse primer complementarity
 Primer-dimer formation between different primers
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Primer Length vs Purity
 Most oligonucleotide synthesis reactions are
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only 98% efficient.
Each time a base is added, only 98% of the
oligos will receive the base.
As length increases, so does the probability
that a primer will be missing a base
Critical in mutagenesis or cloning reactions.
Purification by HPLC or PAGE is
recommended in some cases.
Primer Length vs Purity
Oligonucleotide length
Percent with correct sequence
10 bases
(0.98)10 = 81.7%
20 bases
(0.98)20 = 66.7%
30 bases
(0.98)30 = 54.6%
40 bases
(0.98)40 = 44.6%
Melting Temperature
 Oligonucleotide Factors:
 Primer length
 GC content i.e. Overall Sequence
 Sequence order due to stacking forces:
 nearest neighbour analysis
 Reaction Conditions:
 Salt concentration
 Primer concentration
 Presence of additives in reaction;
 e.g. formamide, DMSO, betaine, glycerol
Relative absorbance at
260nm
Hyperchromic shift & Tm
1.5
ssDNA
1.4
1.3
50% denatured
1.2
1.1
dsDNA
1
40
50
Melting temperature
60
70
80
Temperature (°C)
Experimental determination of DNA melting temperature
90
Relative absorbance at
260nm
Melting Temperature
vs
Annealing Temperature
1.5
ssDNA
1.4
1.3
Annealing
temperatures
1.2
1.1
dsDNA
1
40
50
Melting temperature
60
70
Temperature (°C)
80
90
Mismatched Bases
Mismatched bases
Bonding between neighbouring bases is weakened by the mismatch.
Therefore, the melting temperature is lowered
General Rules for PCR Primers
Innis & Gelfand 1990
1.
2.
3.
4.
5.
6.
7.
Length : 17-28 bases
G+C content : 50-60%
GC clamp: terminal G, C, GC or CG
Primer Tm : 55° - 80°C
Avoid 3’-complementarity
Avoid internal self-complementarity
Avoid runs of 3 or more Gs or Cs near ends
Steps of PCR
 DENATURATION
 PRIMER ANNEALING
 PRIMER EXTENSION BY POLYMERASE
20 to 21
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21 to 22
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22 to 23
DNA Denaturation
5’
3’
ssDNA
Complementary strands
3’
5’
Primer annealing
Annealing & Primer extension
Primer
Polymerase
Template
Primer Design
Using computers to get primers
that will work well
Why use computers?
 Comparison of candidate primer sequence with
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repeat sequences from the species of interest
Nearest-neighbour Tm calculations
Primer self-complementarity analysis
Identification of potential primer-dimer formation
Analysis of large numbers of forward and reverse
primer combinations to find a pair that fit the
desired criteria for target sequence, product size,
primer Tm, etc.
PC based software
 Commercial packages:
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iOligo
DNA Star
PCR Help! (free demo)
Oligo (free demo)
Primer Premier (free demo)
 Free software:
 PerlPrimer
 Oligos
 GeneTool Lite (no longer supported)
Terminology
Forward primer
Target: sequence to be included
between primers
Template: (genomic DNA or cDNA)
Reverse primer
Amplicon:
resulting PCR product
ANGIS Biomanager
 GCG Prime
 Basic selection of oligonucleotide primers for PCR and
sequencing
 CodeHop
 designs a pool of primers containing all possible 11- or
12-mers for the 3' degenerate core region and having
the most probable nucleotide predicted for each
position in the 5' non-degenerate clamp region
 Primer3
“Primer3”
 http://frodo.wi.mit.edu/
 Primer3 picks primers for PCR reactions, according
to the conditions specified by the user.
 Primer3 considers things like
 melting temperature
 concentrations of various solutions in PCR reactions
 primer bending and folding
 Can also pick probes according to specified
parameters
 Variants: e.g. PrimerQuest, with graphic output
 http://scitools.idtdna.com/Primerquest/
“Exon Primer”
 http://ihg.gsf.de/ihg/ExonPrimer.html
 helps to design intronic primers for the PCR
amplification of exons
 needs a cDNA and the corresponding
genomic sequence as input
 can avoid primers to be positioned across
SNPs, using genomic sequence where SNPs
are masked by N’s in input genomic
sequence
“Exon Primer”
SNPs
Multiple Targets: gene exons
Template: genomic DNA
Single amplicon for
small exons/introns
Multiple Amplicons
Overlapping amplicons
for large exons
“CODEHOP”
 http://blocks.fhcrc.org/blocks/codehop.html
 COnsensus-DEgenerate Hybrid
Oligonucleotide Primers
 PCR primers designed from protein multiple
sequence alignments
 Amino acid alignments must be in Blocks
Database format
 Intended for cases where the protein
sequences are distant from each other and
degenerate primers are needed
“POLAND”
 http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html
 Calculates the thermal denaturation profile of
double-stranded RNA, DNA or RNA/DNAhybrids based on sequence input and
parameter settings
e.g. Sequence: 70 44r
CGCCAGCTTGGTCCGAGCTCGGATCCACTAGCTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTACCTGG
PerlPrimer
 http://perlprimer.sourceforge.net/ for downloading
 Free open source standalone
 Runs in Windows, Linux, MacOS
 Features:
 Calculation of possible primer-dimers
 Retrieval of genomic or cDNA sequences from Ensembl (including
both sequences automatically for Q-PCR)
 Ability to BLAST search primers using the NCBI server
 Results can be saved or optionally exported in a tab-delimited format
that is compatible with most spreadsheet applications.
 ORF and CpG island detection algorithms
 Ability to add cloning sequences to primers, automatically adjusted to
be in-frame
 Q-PCR primer design without manual intron-exon boundary entry
PerlPrimer
Other Resources
 NCBI: http://www.ncbi.nlm.nih.gov
 BLAST
 Entrez
 Genome Browser @ UCSC
 http://genome.ucsc.edu/
 Genome Browser
Human
Mouse
Rat
Chimp
 In-Silico PCR
 Blat search
 SNPs
Dog
Chicken
Fugu
C. elegans
Drosophila
S. cerevisiae
Special Applications
Modified Oligonucleotides
&
Special Primers
Degenerate Primers
 Mixed oligos
 e.g. actgattc[gc]tgct[atc]
 Nucleotides can be in unequal ratios
 Increased degeneracy means concentration of the
individual primers decreases
 Deoxyinosine (dI)
 dI at degenerate positions rather than use mixed oligos
 dI base-pairs with any other base, effectively giving a
four-fold degeneracy at any position in the oligo where it
is present
 Degeneracies obviously reduce the specificity
Autosticky PCR
 “dSpacer” protected tetrahydrofuran
phosphoramidite
 For inclusion of abasic sites in an oligo
 Abasic sites cause stalling of DNA polymerases
 Can therefore be used to create 5’-overhangs in
PCR products; “autosticky-PCR”
 Overhangs capable of annealing with restriction
enzyme generated 5’-overhangs
 Chemical 5’-phosphorylation recommended
Real Time PCR
 Considerations for primer design
 Smaller amplicon = higher efficiency
 amplicon ideally < 150 bp; maximum 400 bp
 Amplifying gDNA or cDNA
 gDNA: primers that are intron-specific
 cDNA: primers spanning exon-exon boundaries of
spliced transcript
 Avoid a 3'-end T as this has a greater tolerance
of mismatch
 Primer length: 18–30 nucleotides
Taqman Probes
 Select the probe first and design the primers as
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close as possible to the probe without overlapping it
Tm should be 68°–70°C
No G on the 5´ end
Select the strand that gives the probe more C than
G bases
Avoid runs of an identical nucleotide. This is
especially true for guanine, where runs of four or
more Gs should be avoided
Fluorophore to quencher: optimally 6-14 bases
apart
 Internally positioned quencher increases probe sensitivity
Summary
 Oligo synthesis services that design Q-PCR
primers and probes and guarantee them
 Many useful commercial programmes
 Multiple free tools for designing primers
 PerlPrimer (Desktop computer) : simple
 Primer3 (Web) : highly customisable
 CODEHOP (Web) : for degenerate primers
 Always check your primer sequence!
 Many published primers contain serious errors!
The End
 That link again:
http://users.bigpond.net.au/albert/primers.htm