1. Primer Design and Computer Program Sean Tsai ©2008, National Cheng Kung University Medical College

2. Outlines What is a primer? Does it really matter? Principles of Primer Design Can I trust my gut feeling? What should I do?

3. 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. PCRDNA sequencing

4. After n-th cycle N0N0 N 0 x n N 0 x (1+Y) n-1 Cycle 1 Cycle 2 Cycle 3 Target sequence N 0 copies of template DNA

5. Does it Really Matter? Asuccessful PCR is determined primarily by the quality of primer chosen. Examples:Examples: --- A single base mismatch at the 3’ end of the primer --- Difference of primer Tm --- Too many homology sequences in the mRNA transcripts 1.Story of monkey progesterone receptor 3.Story of human gonadotropin releasing hormone (GnRH) 2.Story of bovine prostaglandin E2 receptor EP3 subtype

6. Principles of Primer Design Use the right sequencesUse 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: Mg 2+, DNA, dNTP concentrations andOther factors: Mg 2+, DNA, dNTP concentrations and

7. Principles of Primer Design Use the right sequenceUse the right sequence Beef, pork, chicken, mouse, or rat? Species specific Yes No Consider the length of PCR product Find consensus sequences between different species Sequence Comparison

8. Principles 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, Cross intron

9. Principles of Primer Design Proper length of primerProper length of primer Usually: 18 - 22 bases Too short Too long Special considerations: Linkers, Restriction enzyme sites, Complementary to specific sequences

10. Principles of Primer Design Suitable annealing temperatureSuitable annealing temperature Depends on primer length and GC content GC content: 45-60% Tm: 55 - 61°C Difference in Tm: =< 2°C

11. Principles of Primer Design (I) A. Avoid hairpin and stem-loop formation B. Minimize primer self-annealing

12. D. Other factors: Mg 2+, DNA, dNTP concentrations Principles of Primer Design (II) C. Minimizing primer-primer annealing

13. Can I trust my gut feeling? Eyes vs. computer programEyes vs. computer program

14. @#*x%# How about pirating other persons primer sequences?

15. What should I do? Find help from primer designing software!Find help from primer 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)

18. Use Oligo 6 to select primer

19. What should I do? Design your primer on web siteDesign your primer on web site Primer 3: http://frodo.wi.mit.edu/ http://frodo.wi.mit.edu/ Vector NTI: http://www.binfo.ncku.edu.t w/nti/ http://www.binfo.ncku.edu.t w/nti/ http://www.binfo.ncku.edu.t w/nti/

20. Primer3

23. Primer3 Output

26. Primer Design 首先先將欲設計 Primer 的 region 圈選出來  再來點選 Analyzes  Primer Design  Find PCR Primers

27. 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

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

29. Design Sequencing primer 首先先將欲設計 Primer 的 region 圈選出來  再來點選 Analyzes  Primer Design  Sequenceing Primers

30. 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

31. How to analyze your primer ? 在 PCR primer result 中按右鍵  選擇 Analyzes 分析一

32. 按下箭頭所指之處 Analyzes  結果會顯示在框框處

33. 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

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

35. 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 )

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

37. 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

38. 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

39. Real-Time PCR Primers Most frequently used machines: ABI Model 7900 and Roche LightCyclerMost frequently used machines: ABI Model 7900 and Roche LightCycler ABI7900: Primer Express (MAC, v1.5 and PC, v2.0)ABI7900: Primer Express (MAC, v1.5 and PC, v2.0) LightCycler: Intrinsic softwareLightCycler: Intrinsic software

40. Primer Express v 2.0

42. Maoa.txt

43. Parameters

44. Start Designing primers

45. Maoa.txt

50. Sequence Input Type in a SequenceType in a Sequence Copy a Sequence from any file and paste it into the LC-PDS sequence windowCopy a Sequence from any file and paste it into the LC-PDS sequence windowNote: allowed characters:A, C, G, T U, if inserting a RNA sequence N, for any possible nucleotide all other characters are disabled minimum sequence length:160 nucleotides maximum sequence length:100,000 nucleotides maximum fragment for analysis:10,000 nucleotides

51. Sequence Input Import a SequenceImport a Sequence – from sequence database files in the –GenBank sequence file format e.g. from http://www.ncbi.nlm.nih.gov –EMBL sequence file format e.g. from http://www.ebi.ac.uk –FASTA sequence file format (used by many sequence alignment and homology search software)

52. Probe Design Software -Entry Screen

53. LightCycler Probe Design Software -entry fields Desired Primer and Probe Tm Minimum Amplicon Size Pr i mer and Probe Concentration

54. LightCycler Probe Design Software - entry fields, continued Roche Buffers Stored in Software Option to Add/Delete Custom Buffer Concentration of Essential Buffer Components

55. LightCycler Probe Design Software - Global Analysis Screen

56. LightCycler Probe Design Software - Primer Probe Sets Screen

57. Homework: Design a primer pair for human hypoxia-inducible factor 1 First: get the sequence from genebank using the methods you learned from this class beforeFirst: get the sequence from genebank using the methods you learned from this class before Second: Use Primer 3 to do it one more timeSecond: Use Primer 3 to do it one more time Third: Design a pair of primers for real time RT-PCRThird: Design a pair of primers for real time RT-PCR And Finally: choose the best primer set of your own and explain why.And Finally: choose the best primer set of your own and explain why.

58. Human hypoxia-inducible factor 1 mRNA NM_001530, NM_181054, AB073325, AB073325 Genomic DNA: Exon 15: AF050127 Accession number: ***Due Time: by midnight of Oct. 5, 2008