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)

Use Oligo 6 to select primerUse Oligo 6 to select primer

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/

Primer3Primer3

Primer3 OutputPrimer3 Output

Primer Design Primer Design

首先先將欲設計 Primer 的 region 圈選出來再來點選 AnalyzesPrimer Design Find PCR Primers

此時出現一新視窗根據個人需求更改參數值按框框所指之處會出現進階選項 按箭頭所指之處可將 Primer 在 5 端加上 Restriction site

結果會顯示在左邊 Text pane 內 , 可自行挑選喜歡的 Primer

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

框框所指之處為更改 sequencing region 的長短再按下 OK 結果ㄧ樣會顯示在左邊

Primer 參數更改之處

How to analyze your primer ?How to analyze your primer ?

在 PCR primer result 中按右鍵選擇 Analyzes

分析一

按下箭頭所指之處 Analyzes 結果會顯示在框框處

分析二 ( 此方法適用於分析 Paired Primer or oligo)

在 PCR primer result 中按右鍵選擇 Add To Oligo List

將 Primer 名稱命名按下 確定 ( 同樣也把 Reverse 的 Primer 也加入 )

接著打開 Oligo List 按下框框所指之鍵但有時會看不到 List 此時將箭頭所指之處往下拉 ( 綠色線條之處 )

接著點選欲分析之 Primer 按下 Analyzes 此時只分析單一 Primer

也可同時分析 Primer 先點選其中之一 , 再按 Shift 此時可同時選擇兩個按下 Duplexes 此時會出現 Oligo Duplexes 視窗按下 Analyzes

此時結果會顯示在框框所指之處顯示這對 Primer 會產生多少種的 Primer Dimer

下一個

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

Primer Express v 2.0Primer Express v 2.0

Maoa.txt

ParametersParameters

Start Designing primersStart Designing primers

Maoa.txt

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)

Probe Design Software -Entry ScreenProbe Design Software -Entry Screen

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