2. Primer Design and Computer Program Does it really matter? Principles of Primer Design Can I trust my gut feeling? What should I do? Sean Tsai ©1999, National Cheng Kung University

3. Does it Really Matter? A successful PCR is determined primarily by the quality of primer chosen. 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

4. Principles of Primer Design Use the right sequences Optimal length of PCR product Proper length of primer Suitable annealing temperature Avoid hairpin and stem-loop formation Minimize primer self-annealing Other factors: Mg 2+, DNA, dNTP concentrations and

5. Principles of Primer Design Use 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

6. 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

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

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

9. Example 471 TCGTTGGATCCCACGGCCAGCCAGCTGCAGGTGCTGCTGGGCGTCCCTGT 520 | ||||| | ||| || | ||| |||| | ||||| || ||| 473 GCCTTGGACCACACAGCTGACAGGCTACAGGCAATCCTGGGTGTTCCTTG 522..... 521 GAAGGAGGGAGACTGCACCTCCCGGCTGGACGGACATAAGGTCCTCACTG 570 |||||| |||||||||||||||||||||||||||||||||| ||| 523 GAAGGACAAGAACTGCACCTCCCGGCTGGACGGACACAAGGTCCTGTCTG 572..... 571 CCCTGCAGGCTGTTCAGGGCTTGCTGGTCACCCAGGGTGGAAGCAGCAGC 620 ||||||||||||| |||||| |||| || ||||||| | ||| 573 CCCTGCAGGCTGTACAGGGCCTGCTAGTGGCCCAGGGCAGGGCTGATAGC 622..... 621 CAGACACCCCTGCTACAGTCCACCGTGGTGGGCCTCTTCACTGCCCCAGG 670 ||| | | ||||| | |||||| ||||||||| | ||||| |||||||| 623 CAGGCCCAGCTGCTGCTGTCCACGGTGGTGGGCGTGTTCACAGCCCCAGG 672..... 671 CTTGCGCCTAAAACAGCCATTTGTTGAGAGCTTGGGTCCCTTCACCCCCG 720 | ||| ||| || ||||| ||||| || || ||| || || ||||| | 673 CCTGCACCTGAAGCAGCCGTTTGTGCAGGGCCTGGCTCTCTATACCCCTG 722

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

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

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

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

14. What should I do? Find help from primer designing software! Oligo Synthesis Program: Hillier, L. and Green, P. PCR Methods and Applications, 1:124-128. (1991) Oligo 6 (by Wojciech Rychlik): commercially available (National Sciences, Inc) Vector NTI: from MDBio, Inc. http://www.mdbio.neto.net/vnti.htm

20. Use Oligo 6 to select primer

21. What should I do? Design your primer on web site GCG: telnet://gcg.nhri.org.tw Primer 3: http://www- genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi http://www- genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi Seg Web: http://gcg.nhri.org.tw:8003/gcg- bin/seqweb.cgi http://gcg.nhri.org.tw:8003/gcg- bin/seqweb.cgi

22. Primer3

24. Primer3 Output

26. % prime Prime of what sequence ? ggamma.seq Begin (* 1 *) ? End (* 1700 *) ? 500 Minimum primer length (* 18 *) ? Maximum primer length (* 22 *) ? Minimum product length (* 100 *) ? Maximum product length (* 300 *) ? What should I call the output file name (* ggamma.prime *) ? This program can display the primer binding sites graphically. Do you want to: A) Plot to a FIGURE file called "prime.figure" B) Plot graphics on LaserWriter attached to /dev/tty10 C) Suppress the plot Please choose one (* A *): Searching for forward primers.......................................... Searching for reverse primers......................................... Selecting primer pairs.............................................................................................................. GCG’s Primer Design Command (I)

27. PRIME of: ggamma.seq ck: 3814 from: 1 to: 500 September 27, 1996 11:21 INPUT SUMMARY ------------- Input sequence: GGamma.Seq Primer constraints: primer size: 18 - 22 primer 3' clamp: S primer sequence ambiguity: NOT ALLOWED primer GC content: 40.0 - 55.0% primer -: 50.0 - 65.0 degrees Celsius primer self-annealing... 3' end: < 8 (weight: 2.0) total: < 14 (weight: 1.0) unique primer binding sites: required primer-template and primer-repeat annealing... 3' end: ignored total: ignored repeated sequences screened: none specified Product constraints: product length: 100 - 300 product GC content: 40.0 - 55.0 product Tm: 70.0 - 95.0 degrees Celsius duplicate primer endpoints: NOT ALLOWED difference in primer Tm: < 2.0 degrees Celsius primer-primer annealing... 3' end: < 8 (weight: 2.0) total: < 14 (weight: 1.0) PRIMER SUMMARY -------------- forward reverse Number of primers considered: 1387 1386 Number of primers rejected for primer 3' clamp: 227 225 primer sequence ambiguity: 0 0 primer GC content: 623 633 primer Tm: 67 72 non-unique binding sites: 0 0 primer self-annealing: 54 52 primer-template annealing: 0 0 primer-repeat annealing: 0 0 Number of primers accepted: 416 404 GCG’s Primer Design Command (II)

28. PRODUCT SUMMARY --------------- Number of products considered: 168064 Number of products rejected for... product length: 124863 product GC content: 1353 product Tm: 0 product position: 0 duplicate primer endpoints: 15020 difference in primer Tm: 17485 primer-primer annealing: 7690 Number of products accepted: 1653 Number of products saved: 25 Output for GCG’s Prime (I)

29. Output of GCG’s Prime (II) Product: 1 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS ------- 5' 3' forward primer (19-mer): 13 TCAGCAGTTCCACACACTC 31 reverse primer (18-mer): 159 TTCTCCTCCAGCATCTTC 142 forward reverse primer %GC: 52.6 50.0 primer Tm (degrees Celsius): 55.5 53.5 PRODUCT product length: 147 product %GC: 51.0 product Tm: 76.8 degrees Celsius difference in primer Tm: 2.0 degrees Celsius annealing score: 37 optimal annealing temperature: 54.9 degrees Celsius

30. Design a primer pair for human LH receptor First: get the sequence from genebank using the methods you learned from this class before Second: use GCG command mode to do it Third: Use Primer 3 to do it one more time

31. Human LH receptor mRNA S57793, M73746, M63108, E05678 Genomic DNA: Exon 5: X84760 Accession number: