Primer Design 2013.pdf

7/27/2019 Primer Design 2013.pdf

1/58

Primer DesignDESIGN OF OLIGONUCLEOTIDE PRIMERS FORBASIC PCRJ E R E M Y G . V I C E N C I O

D E PA R T M E N T O F B I O L O G Y

C O L L E G E O F A R T S A N D S C I E N C E S

U N I V E R S I T Y O F T H E P H I L I P P I N E S M A N I L A


2/58

Before you start designing primers

find and use the right resources!o What are the primers for?

o General purposeamplification?

o SNPs detection/validation

o Methylation study?

o Real-time PCR?

o Microarray probes?

o Degenerate PCR?

o Multiplex PCR?

o What do you have to begin

with?

o Single DNA/protein

sequence?

o Multiple DNA/protein

sequence files?

o

GenBank ID/Gene ID/GeneSymbol/rsSNP ID?


3/58

After you have designed your primers

Consider a second opinion!o Several different software are available for designing

primers

o Various software may differ significantly in:

o Concrete and overall approaches

o Designing criteria and default settings

o Comprehensiveness

o Usability, accessibility, and speed

o Consider a second opinion when

o You are new to such a design task/application

o You dont have a lot of confidence in the initial result


4/58

Question 1What is the chief goal of primer design?

A. Specificity

B. Sensitivity


5/58

Selecting PCR Primerso Analysis of the target gene for potential primingsites

o free of homopolymeric tracts

o have no obvious tendency to form secondarystructures

o not self-complementary

o have no significant homology with other sequences

on either strand of the target genome

o Creation of lists of possible forward and reverseprimers


6/58

Selecting PCR Primerso Selection of well-matched pairs of forward and

reverse primerso similar in G+C content

o must generate an amplified product of the

appropriate size and base composition

o Refining the length and/or placement of theoligonucleotides


7/58

Properties of OligonucleotidesThat Influence the Efficiency ofAmplification


8/58

Base Compositiono G+C content should be somewhere between 40%

and 60%, with an even distribution of all fourbases along the length of the primero avoid polypurine tracts or polypyrimidine tracts

o avoid long runs of a single base (max 4 bases)

o avoid dinucleotide repeats (max 4 repeats)


9/58

Lengtho Primer length determines the specificity and significantly

affects its annealing to the template

o Too short low specificity, resulting in non-specific

amplificationo Too long decrease the template-binding efficiency at normal

annealing temperature due to the higher probability offorming secondary structures such as hairpins

o

Optimal primer lengtho 18-25 nucleotides long for general applications

o 30-35 nucleotides for multiplex PCR

o Members of a primer pair should not differ in length by >3 bp.


10/58

Lengtho Optimal amplicon size

o 300-1000 bp for general applications, avoid >3 kb

o

50-150 bp for real-time PCR, avoid >400 bp


11/58

Repeated and Self-

Complementary Sequenceso No inverted repeat sequences or self-

complementary sequences >3 bp in length

should be present.

o If primers can anneal to themselves, or anneal to

each other than anneal to the template, the PCR

efficiency will be decreased dramatically. They

shall be avoided.


12/58

Secondary structures

Hairpino Formed via intra-molecular

interactions

o

Negatively affect primer-template binding, lead to pooror no amplification

o Acceptable G (free energyrequired to break the structure):>-2 kcal/mol for 3end hairpin;>-3 kcal/mol for internal hairpin


13/58


Self-dimer (homodimer)o Formed by inter-

molecular interactions

between the two same

primers

o Acceptable G: >-5kcal/mol for 3 end self-

dimer; >-6 kcal/mol forinternal self-dimer


14/58

Complementarity between

members of a primer pairo The 3 terminal sequences of one primer should

not be able to bind to any other site on the other

primer.

o Formation of primer dimers can be reduced by

o careful primer design

o use of hot start or touchdown PCR

o use of specially formulated DNA polymerases


15/58


Cross-dimer (heterodimer)o Formed by inter-

molecular interactionsbetween the sense and

antisense primers

o Acceptable G: >-5kcal/mol for 3 end

cross-dimer; >-6kcal/mol for internalcross-dimer


16/58

What is a primer dimer?o An unwanted extension

product

o Results from primers

annealing tothemselves, or eachother, at 3 ends

o Extended primers areno longer available toprime target for PCR

atcggactatcga

gctatacttatggcca

atcggactatcgatatgaataccgga

tagcctgatagctatacttatggcca


17/58

Melting Temperatures (T

m)o Tm is the temperature at which 50% of the DNA

duplex dissociates to become single-stranded

o Determined by primer length, base composition, and

concentration

o Also affected by the salt concentration of the PCR reaction

mix


18/58


m)o Wallace rule: Tm (in C) = 2(A+T)+4(G+C)

o can be used to calculate the melting temperature for perfect

duplexes 15-20 nucleotides in length in solvents of high ionic

strength (e.g., 1 M NaCl):

o Baldino algorithmTm (in C) = 81.5C + 16.6 (log10[K+]) + 0.41(%[G+C]) (675/n)where n is the number of bases in the oligonucleotideo predicts reasonably well the melting temperature of

oligonucleotides, 14-70 nucleotides in length, in cation

concentrations of 0.4 M or less:


19/58


m)o Should not differ by >5C between members of a

primer pair

o The Tm of the amplified product should not differfrom the Tm values of the primer pairs by >10C


20/58


m)o Optimal Tm

o 52C to 60C

o Tm

above 65C should be avoided because of the

potential for secondary annealing

o Higher Tm is recommended for amplifying high GC

content targets


21/58

3 Terminio If possible, the 3 base of each primer should be

G or C.

o However, primers with a .NNCG or .NNGC

sequence at their 3 termini are not recommended

since this promotes the formation of hairpin

structures and may generate primer dimers.

o GC Clamp refers to the presence of G or Cwithin the last 5 bases from the 3 end of primers

o Essential for preventing mis-priming and enhancing

specific primer-template binding


22/58

Adding restriction sites, bacteriophagepromoters, and other sequences to the

5 termini of primerso In general, the presence of additional sequences

does not significantly affect annealing of the

oligonucleotide to its target DNA.

o Restriction sites: the primer should be extendedby at least three additional nucleotides beyond

the recognition sequence of the restriction

enzyme.


23/58

Placement of priming siteso Various constraints on the placement of priming

sites:

o mutations

o restriction sites

o coding sequences

o microsatellites

o cis-acting elements

o Use forward and reverse primers that bind todifferent exons when designing primers for useon cDNA templates.


24/58

Cross-homologyo Cross homology may become a problem when PCR template

is genomic DNA or consists of mixed gene fragments.

o BLASTing PCR primers against the NCBI non-redundant

sequence database is a common way to avoid designingprimers that may amplify non-targeted homologous regions.

o Use primers spanning intron-exon boundaries to avoid non-specific amplification of gDNA due to cDNA contamination.

o Use primers spanning exon-exon boundaries to avoid non-specific amplification cDNA due to gDNA contamination.


25/58

Annealing temperature (Ta)

o Ta vs. Tmo Ta is determined by the Tm of both primers and amplicons:

optimal Ta = 0.3 x Tm (primer) + 0.7 x Tm (amplicon) 25o General rule: Ta is 5C lower than Tmo Higher Ta enhances specific amplification but may reduce

yield


26/58

Test yourunderstandingEVALUATE WHETHER TH E FOLLOWING P RIMERSMEET THE GUIDELINES FOR EACH SPECIFIEDCRITERION


27/58

Question 2

o Length

5 ACT GCC TCG ACT ACT TAC GC 3


28/58

Question 3

o Length

Forward: 5 GCT TCA ACG GAC CAT TGC 3Reverse: 5 CTT ACG ACT TCC ACT TCC GCA C 3


29/58

Question 4

o Base composition

5 GCCACGTCGCGCATGCGC 3


30/58

Question 5

o Base Composition

5 ACACACACTTTTGCC 3


31/58

Question 6

o Self-complementary sequences

5 GCTTCACGGATCTGAAGC 3


32/58

Question 7

o Hairpins

5 ACGCTCTCCACGAGTCACGC 3


33/58

Question 8

o Homodimers

5 GCGTTAGGCACGAATCACGC 3


34/58

Question 9

o Heterodimers

Forward:5 GCGTTAGGCACGAATCACGC 3

Reverse:

5 GCGTCAGAACCGTACGAGCG 3


35/58

Question 10

o Melting Temperature

5 ATTGAATCTACTACTTACGC 3Tm: 50.7C


36/58

Question 11

o Melting Temperature

Forward: 5 GATTTAAGCACGAATCACGC 3Tm: 54.7C

Reverse: 5 TAGTCAGCATCGTATGAGCG 3Tm: 58.0C

Amplified Product: 851 bp, Tm: 64.0C


37/58

Question 12

o 3 Terminus

5 GATTTAAGCACGAATCACGC 3


38/58

Computer-Assisted Designof Oligonucleotide Primers


39/58

Primer3

o Web-based tool for primer design

http://frodo.wi.mit.edu/o Example gene: GFP5 Green Fluorescent Protein

o GenBank: U87973.1
http://frodo.wi.mit.edu/http://frodo.wi.mit.edu/


40/58

Primer3

Enter sequence

Pick Primers


41/58

Primer Size

Primer Tm

Complementarity

Primer3 Advanced Controls


42/58

Primer3 Output

Details:-Start

-Length-Tm-% GC-Sequence

Where they bind:


43/58

Primer3 Output


44/58

Primer Evaluation

o OligoAnalyzer 3.1 (web-based)

http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/
http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/


45/58


46/58

OligoAnalyzer 3.1: Analyze


47/58

OligoAnalyzer 3.1: Hairpin


48/58

OligoAnalyzer 3.1: Homodimer


49/58

OligoAnalyzer 3.1: Heterodimer


50/58

AmplifX

o Standalone desktop software


51/58

AmplifX


52/58

AmplifX


53/58

OligoAnalyzer 1.5



54/58

OligoAnalyzer 1.5


55/58

OligoExplorer 1.5



56/58

OligoExplorer 1.5


57/58

Primer-BLAST

o Web-based tool

o Provides primers specific to the PCR template

sequence.

o Includes primer pair specificity checking against a

selected database.


58/58

Primer-BLAST

Primer Design 2013.pdf

Documents

Transcript of Primer Design 2013.pdf