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Primer Design and Primer Design and Computer ProgramComputer Program
Sean Tsai ©2008, National Cheng Kung University Medical College
OutlinesOutlines
What is a primer?What is a primer?
Does it really matter?Does it really matter?
Principles of Primer DesignPrinciples of Primer Design
Can I trust my gut feeling? Can I trust my gut feeling?
What should I do?What should I do?
What is a primer?What is a primer?
A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.
After n-th cycle
N0 N0 x n N0 x (1+Y)n-1
Cycle 1
Cycle 2
Cycle 3
Target sequence
N0 copies of template DNA
Does it Really Matter?Does it Really Matter?
• A successful PCR is determined primarily by the A successful PCR is determined primarily by the quality of primer chosen.quality of primer chosen.
• Examples:Examples:
--- --- A single base mismatch at the 3’ end of the primerA single base mismatch at the 3’ end of the primer
--- --- Difference of primer TmDifference of primer Tm
--- --- Too many homology sequences in the mRNA Too many homology sequences in the mRNA transcriptstranscripts
1.1. Story of monkey progesterone receptorStory of monkey progesterone receptor
3.3. Story of human gonadotropin releasing Story of human gonadotropin releasing hormone (GnRH)hormone (GnRH)
2.2. Story of bovine prostaglandin E2 receptor EP3 Story of bovine prostaglandin E2 receptor EP3 subtypesubtype
Principles of Primer DesignPrinciples of Primer Design
• Use the right sequences Use the right sequences • Optimal length of PCR productOptimal length of PCR product• Proper length of primerProper length of primer• Suitable annealing temperatureSuitable annealing temperature• Avoid hairpin and stem-loop formationAvoid hairpin and stem-loop formation• Minimize primer self-annealingMinimize primer self-annealing
• Other factors: Other factors: MgMg2+2+, DNA, dNTP concentrations and, DNA, dNTP concentrations and
Principles of Primer DesignPrinciples of Primer Design
• Use the right sequenceUse the right sequence
BeefBeef, pork, , pork, chickenchicken, , mousemouse, or , or ratrat??
Species specificYesYes
NoNo
Consider the length of PCR product
Find consensus sequences between different species
Sequence Comparison
Principles of Primer DesignPrinciples of Primer Design
• Optimal length of PCR product
B. For probing:150 - 300 bp
A. Cloning cDNA: Full length
C. Checking Polymorphism: 100 - 300 bp
D. Quantification:300 - 500 bp
E. General: 300 - 1000 bp
Proper restriction sites,Proper restriction sites, Cross intronCross intron
Principles of Primer DesignPrinciples of Primer Design
• Proper length of primerProper length of primer
Usually: 18 - 22 basesUsually: 18 - 22 bases
Too shortToo short
Too longToo long
Special considerations:Special considerations:
Linkers, Restriction enzyme sites, Complementary to specific sequences
Principles of Primer DesignPrinciples of Primer Design
• Suitable annealing temperatureSuitable annealing temperature
Depends on primer length and GC contentDepends on primer length and GC content
GC content:GC content: 45-60%45-60%
Tm:Tm:55 - 61°55 - 61°CC
Difference in Tm:Difference in Tm: =< 2°=< 2°CC
Principles of Primer Design (I)Principles of Primer Design (I)
A. Avoid hairpin and stem-loop A. Avoid hairpin and stem-loop formationformation
Current Oligo, 20-mer [68]:Current+ Oligo: the most stable 3'-dimer: 2 bp, -1.9 kcal/mol
5' CCAGTCGTTACAAACTGACA 3'
3' ACAGTCAAACATTGCTGACC 5' :: :: | |
Current- Oligo: no 3'-terminal dimer formation
Current+ Oligo: the most stable dimer overall: 4 bp, -4.8 kcal/mol
5' CCAGTCGTTACAAACTGACA 3'
3' ACAGTCAAACATTGCTGACC 5' :: :: :: :: :: :: | | | |
Hairpin: ²G = -0.7 kcal/mol, Loop = 8 nt, Tm = 41°
5' CCAGTCGTTA
3' ACAGTCAAACA| | | |
B. Minimize primer self-annealingB. Minimize primer self-annealingCurrent Oligo, 20-mer [16]:Current+ Oligo: no 3'-terminal dimer formation
Current- Oligo: the most stable 3'-dimer: 6 bp, -7.8 kcal/mol
5' GTGATGGCTAGGATATTAAT 3'
3' TAATTATAGGATCGGTAGTG 5' | | | | | |
Current+ Oligo: the most stable dimer overall: 6 bp, -7.8 kcal/mol
5' ATTAATATCCTAGCCATCAC 3'
3' CACTACCGATCCTATAATTA 5' | | | | | |
No hairpin stems 3 bp
Primer positions: U 35 L 106Upper/Lower: the most stable 3'-dimer: 4 bp, -6.3 kcal/mol
5' CGGGAAATGTGACTGTCCTC 3'
3' ACGTTGGAGAGGAAACGTCT 5' | | | |
Upper/Lower: the most stable 3'-dimer: 2 bp, -1.9 kcal/mol
5' CGGGAAATGTGACTGTCCTC 3'
3' ACGTTGGAGAGGAAACGTCT 5' :: :: :: | |
Upper/Lower: the most stable dimer overall: 4 bp, -6.3 kcal/mol
5' CGGGAAATGTGACTGTCCTC 3'
3' ACGTTGGAGAGGAAACGTCT 5' :: :: | | | |
D. Other factors: MgD. Other factors: Mg2+2+, DNA, dNTP , DNA, dNTP concentrationsconcentrations
Principles of Primer Design (II)Principles of Primer Design (II)
C. Minimizing primer-primer annealingC. Minimizing primer-primer annealing
Can I trust my gut feeling?Can I trust my gut feeling?
• Eyes vs. computer Eyes vs. computer programprogram
1 CCTGAGTCGG TCTCCAGCAC GCAACGGCCA CCCTGCACCA TGAGCCGGCA
51 GAGTATCTCG CTCCGATTCC CGCTGCTTCT CCTGCTGCTG TCGCCATCCC
101 CCGTCTTCTC AGCGGACCCC GGGGCGCCCG CGCCAGTGAA CCCCTGCTGT
151 TACTATCCAT GCCAGCACCA GGGGATCTGT GTTCGCTTCG GCCTTGACCG
201 CTACCAATGT GACTGCACCC GCACGGGCTA TTCCGGCCCC AACTGCACCA
251 TCCCGGAGAT ATGGACCTGG CTCCGGACGA CTTTGCGGCC CAGCCCCTCT
301 TTCATCCACT TTCTGCTGAC GCACGGGCGC TGGCTTTGGG ATTTTGTCAA
351 TGCCACCTTC ATCCGGGACA CGCTCATGCG TCTGGTACTC ACAGTGCGTT
401 CCAACCTTAT CCCCAGCCCT CCCACCTACA ACATAGCGCA CGATTACATC
451 AGCTGGGAGT CCTTCTCCAA TGTGAGCTAT TATACTCGCA TTCTGCCCTC
501 CGTGCCCCGA GACTGTCCCA CGCCCATGGA CACCAAAGGG AAGAAGCAAT
551 TGCCAGATGC GGAGTTCCTG AGCCGTCGCT TCCTGCTCAG GAGGAAGTTC
601 ATCCCTGACC CTCAAAGCAC CAACCTCATG TTTGCCTTCT TTGCCCAACA
651 CTTCACCCAT CAGTTCTTCA AAACTTCCGG CAAGATGGGT CCTGGCTTCA
701 CCAAGGCGCT GGGCCACGGG GTAGACCTCG GCCACATTTA TGGAGACAAT
751 CTGGAACGTC AGTATCAGCT GCGGCTCTTT AAGGATGGGA AGCTGAAGTA
801 CCAGATGCTC AATGGAGAGG TGTACCCGCC ATCGGTGGAA GAGGCGCCCG
851 TGCTGATGCA CTACCCCCGG GGCATCCCGC CCCAGAGCCA GATGGCTGTG
901 GGCCAGGAGG TGTTTGGGCT GCTTCCTGGA CTCATGCTCT ACGCCACGAT
951 CTGGCTGCGT GAGCACAACC GCGTGTGTGA CCTGCTGAAG GCTGAGCACC
@#*x%#
How about pirating other persons How about pirating other persons primer sequences?primer sequences?
What should I do?What should I do?
• Find help from primer Find help from primer designing software!designing software!
Oligo 6 (by Wojciech Rychlik): commercially available (National Sciences, Inc)
Vector NTI: from Invitrogen, Inc. http://www.informaxinc.com/
Oligo Synthesis Program: Hillier, L. and Green, P. PCR Methods and Applications, 1:124-128. (1991)
What should I do?What should I do?
• Design your primer on Design your primer on web siteweb site
Primer 3:Primer 3: http://frodo.wi.mit.edu/
Vector NTI: Vector NTI: http://www.binfo.ncku.edu.tw/nti/
Design Sequencing primerDesign Sequencing primer
首先先將欲設計 Primer 的 region 圈選出來再來點選 AnalyzesPrimer Design Sequenceing Primers
Real-Time PCR PrimersReal-Time PCR Primers
• Most frequently used machines: ABI Most frequently used machines: ABI Model 7900 and Roche LightCyclerModel 7900 and Roche LightCycler
• ABI7900: Primer Express (MAC, v1.5 and ABI7900: Primer Express (MAC, v1.5 and PC, v2.0)PC, v2.0)
• LightCycler: Intrinsic softwareLightCycler: Intrinsic software
SequenceSequence InputInput
• Type in a SequenceType in a Sequence• Copy a SequenceCopy a Sequence
from any file and paste it into the LC-PDS sequence windowfrom any file and paste it into the LC-PDS sequence window
Note:Note:allowed characters:allowed characters: A, C, G, T A, C, G, T
U, if inserting a RNA sequence U, if inserting a RNA sequence N, for any possible nucleotideN, for any possible nucleotide
all other characters are disabledall other characters are disabled
minimum sequence length:minimum sequence length: 160 nucleotides160 nucleotidesmaximum sequence length:maximum sequence length: 100,000 nucleotides100,000 nucleotidesmaximum fragment for analysis:maximum fragment for analysis: 10,000 nucleotides10,000 nucleotides
Sequence Input Sequence Input
• Import a SequenceImport a Sequence– from sequence database files in thefrom sequence database files in the
– GenBank sequence file formatGenBank sequence file formate.g. from http://www.ncbi.nlm.nih.gove.g. from http://www.ncbi.nlm.nih.gov
– EMBL sequence file formatEMBL sequence file formate.g. from http://www.ebi.ac.uke.g. from http://www.ebi.ac.uk
– FASTA sequence file formatFASTA sequence file format(used by many sequence alignment and homology (used by many sequence alignment and homology search software)search software)
LightCycler Probe Design LightCycler Probe Design Software -entry fieldsSoftware -entry fields
Desired Primer and
Probe Tm
Minimum Amplicon Size
Primer and Probe
Concentration
LightCycler Probe Design Software -LightCycler Probe Design Software -entry fields, entry fields, continuedcontinued
Roche Buffers
Stored in Software
Option to Add/Delete
Custom Buffer
Concentration of
Essential Buffer
Components
LightCycler Probe Design Software -LightCycler Probe Design Software -Global Analysis ScreenGlobal Analysis Screen
LightCycler Probe Design Software -LightCycler Probe Design Software -Primer Probe Sets ScreenPrimer Probe Sets Screen
Homework: Design a primer pair for Homework: Design a primer pair for human hypoxia-inducible factor 1human hypoxia-inducible factor 1
• First:First: get the sequence from get the sequence from genebank using the methods you genebank using the methods you learned from this class beforelearned from this class before
• Second:Second: Use Primer 3 to do it one Use Primer 3 to do it one more time more time
• Third: Third: Design a pair of primers for Design a pair of primers for real time RT-PCRreal time RT-PCR
• And Finally:And Finally: choose the best primer choose the best primer set of your own and explain why.set of your own and explain why.
Human hypoxia-inducible factor 1Human hypoxia-inducible factor 1
mRNA mRNA NM_001530 NM_001530 ,, NM_181054, AB073325, AB073325 NM_181054, AB073325, AB073325
Genomic DNA:Genomic DNA: Exon 15: AF050127Exon 15: AF050127
Accession number:Accession number:
***Due Time: by midnight of Oct. 5, 2008***Due Time: by midnight of Oct. 5, 2008
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