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

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

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: Mg2+, DNA, dNTP concentrations

and

Principles of Primer Design

Use the right sequence

Beef, pork, chicken, mouse, or rat?

Species specificYes

No

Consider the length of PCR product

Find consensus sequences between different species

Sequence Comparison

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

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

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

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

Principles of Primer Design (I)

A. Avoid hairpin and stem-loop formation

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-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: Mg2+, DNA, dNTP concentrations

Principles of Primer Design (II)

C. Minimizing primer-primer annealing

Can I trust my gut feeling?

Eyes vs. computer program

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 primer sequences?

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

PROD_LEN_M IN 100 PROD_LEN_MA X 3700 PROD_GC_M IN 40 PROD_GC_M AX 70 PROD_TM _M IN 65.0 PROD_TM _M AX 95.0 PRIM _GC_M IN 40 PRIM _GC_M AX 60 PRIM _LEN_MI N 18 PRIM _LEN_M AX 22 PRIM _TM_ MI N 55.0 PRIM _TM_ M AX 61.0 PRIM _TM_ DI FF 2.0

AT _SCORE 2.0 CG_SCORE 4.0 WT_AM BIG avg PRIM _SELF_I_ANN 21.0 PRIM _SELF_3_ANN 8.0 PRIM _PRIM _I_ANN 14 .0 PRIM _PRIM _3_ANN 8.0 PRIM _PROD_I_ANN 0.0 PRIM _PROD_3_ANN 0.0 PRIM _OTHER_I_ANN 0.0 PRIM _OTHER_3_ANN 0.0

**********************************************************PRIMERS(Number of N-type sequences found was 38) Sense: Total considered: 110 Rejected based on ambiguity (N): 0 Rejected based on gc_content: 0 Rejected based on primer Tm: 25 Rejected based on self-annealing 21 Rejected based on other-annealing : 0 Number accepted: 64 Antisense: Total considered: 60 Rejected based on ambiguity (N): 0 Rejected based on gc_content: 2 Rejected based on primer Tm: 30 Rejected based on self-annealing: 0 Rejected based on other-annealing : 0 Number accepted: 28PRODUCTS Total considered: 1792 Rejected based on identical endpts: 703 Rejected based on length: 0 Rejected based on primer-primer annealing : 131 Rejected based on primer-product annealing : 0 Rejected based on melting temperature : 0 Rejected based on gc_content: 0 Rejected based on difference in Tm : 824 Number accepted: 134*******************************************************

Primer Pair # 1 5' end 3' end length G+C(%) TmOLIGO1: ...aaCCTCTCCTTTGCAGACTTctg... 111 128 18 50 55.0OLIGO2: ...caCGTTGAGAATCAGGATGGTtaa... 517 499 19 47 55.4 ANNEALING SCORES PRIMER-SELF PRIMER-PRIMER PRIMER-PRODUCT PRIMER-OTHER 3' Internal 3' Internal 3' Internal 3' InternalOLIGO 1: 2.0 12.0 10.0 20.0 16.0 20.0OLIGO 2: 2.0 8.0 6.0 12.0 18.0 22.0 18.0 22.0 PRODUCT Length G+C(%) Tm Total Score 407 50 81.8 52.0

Primer Pair # 2 5' end 3' end length G+C(%) TmOLIGO1: ...aaCCTCTCCTTTGCAGACTTctg... 111 128 18 50 55.0OLIGO2: ...caCGTTGAGAATCAGGATGGTTAaga... 517 497 21 42 56.1 ANNEALING SCORES PRIMER-SELF PRIMER-PRIMER PRIMER-PRODUCT PRIMER-OTHER 3' Internal 3' Internal 3' Internal 3' InternalOLIGO 1: 2.0 12.0 10.0 20.0 16.0 20.0OLIGO 2: 4.0 8.0 4.0 12.0 6.0 22.0 16.0 22.0 PRODUCT Length G+C(%) Tm Total Score 407 50 81.8 52.0

Primer Pair # 3 5' end 3' end length G+C(%) TmOLIGO1: ...aaCCTCTCCTTTGCAGACTTctg... 111 128 18 50 55.0OLIGO2: ...caCGTTGAGAATCAGGATGGTTaag... 517 498 20 44 55.8 ANNEALING SCORES PRIMER-SELF PRIMER-PRIMER PRIMER-PRODUCT PRIMER-OTHER 3' Internal 3' Internal 3' Internal 3' InternalOLIGO 1: 2.0 12.0 10.0 20.0 16.0 20.0OLIGO 2: 4.0 8.0 4.0 12.0 16.0 22.0 16.0 22.0 PRODUCT Length G+C(%) Tm Total Score 407 50 81.8 52.0

Use Oligo 6 to select primer

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.cgiSeg Web: http://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgi

Primer3

Primer3 Output

% 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)

PRIME of: ggamma.seq ck: 3814 from: 1 to: 500 September 27, 1996 11:21

INPUT SUMMARY ------------- Input sequence: GGamma.SeqPrimer 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 ALLOWEDdifference 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)

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)

Output of GCG’s Prime (II)Product: 1

[DNA] = 50.000 nM [salt] = 50.000 mMPRIMERS

-------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

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

Human LH receptor

mRNA S57793, M73746, M63108, E05678

Genomic DNA: Exon 5: X84760

Accession number: