from: Eugene Shpaer e-mail: gene@jmullins.stanford.edu tel. (415) 723-0707 US Mail: Dept. Microbiol, Stanford Univ., CA 94305-5402, USA The program 'primer' is written by Don Faulkner. It helps to find potential mispriming sites (primer sequences should be designed before running the program!). The program allows to give higher weights to matches at the 3' end of the primer, linearly decreasing them towards the 5' end (the default is weight=10 for 3' nucleotide decreasing to 1 at nucleotide # 8 from the 3' end). The program can be used when amplifying *long* fragments from a known sequence. The program is written in "C" and runs on Sun workstation (Unix). To get the program running: %ftp 36.45.0.126 or jmullins.stanford.edu logon: anonymous send your ID as a passwd cd pub set binary get primer.tar .... %tar xvf primer.tar %make primer %primer ... and you should get the sample run, presented below. List of files: -rw-rw-rw- 1 gene 532 Apr 8 18:33 Makefile -rw-rw-rw- 1 gene 1527 Apr 8 17:25 backup.c -rw-rw-rw- 1 gene 14226 Apr 8 17:16 clib.c -rw-rw-rw- 1 gene 6174 May 7 1990 dynamic.c -rw-rw-rw- 1 gene 208 May 7 1990 dynamic.h -rw-rw-rw- 1 gene 2999 Apr 8 17:23 faulkn.h -rw-rw-rw- 1 gene 208 May 7 1990 free.c -rw-rw-rw- 1 gene 5328 May 7 1990 get-matrix.c -rw-rw-rw- 1 gene 580 May 8 1990 hello.c -rw-rw-rw- 1 gene 6679 Jun 5 1990 main.c -rw-rw-rw- 1 gene 245 May 7 1990 matrix-test.c -rw-rw-rw- 1 gene 8609 Apr 8 17:27 name-expander.c -rw-rw-rw- 1 gene 1145 Apr 8 17:25 nread.c -rw-rw-rw- 1 gene 4549 Apr 8 17:16 open_file.c -rw-rw-rw- 1 gene 1629 May 11 1990 position-mat.c -rw-rw-rw- 1 gene 8094 May 11 1990 rdseq.c The file -rw-r--r-- 1 gene 229 Apr 8 18:16 matrix has the following: actg ; this is the character set match = 2 close = match / 2 missm = -match ;a c t g match missm missm missm ;a missm match missm missm ;c missm missm match close ;t missm missm close match ;g These are sample files with several primer sequences and long template: -rw-rw-rw- 1 gene 89 May 11 1990 3_primers -rw-rw-rw- 1 gene 26769 May 11 1990 hxb-inv.seq -rw-r--r-- 1 gene 40 Apr 8 17:31 .primer Sample run is below: %primer ********************* * * * DYNAMIC PRIMERS * * * ********************* Parameters for this search will be taken from the previous one you made OR you can specify new parameters. Weighted search using the Dynamic Programming Algorighm Gap Penalty, or for -2.000000 -> LARGE sequence: Enter name of file containing query sequence, or for hxb-inv.seq -> Locus names in the file : HXB2-INV Enter locus name of query sequence, or for HXB2-INV -> 9719 bases found for locus HXB2-INV in file hxb-inv.seq PRIMER sequence: Enter name of file containing query sequence, or for 3_primers -> Locus names in the file <3_primers>: sbc4 sbc5 sbc6 Enter locus name of query sequence, or for sbc4 -> 19 bases found for locus sbc4 in file 3_primers Locus "HXB2-INV" (len = 9719) against "sbc4" (len = 19) Position weighting matrix: Default is Val=1 from 5' end to position (length-8) linearly increasing to Val=10 at the 3' end of the primer. If you want to CHANGE it enter 'y' Output file: File to write to -> test 2391 maxima #1 score 95.875, 1 gap 4840 AATTTG-TTTTTGTAATTC ****. *.******* 1 CATTTTCCAATGGTAATTC #2 score 95.125, 2 gaps 671 CAGCTGCC--TTGTAAGTC **. *.** *.****.** 1 CATTTTCCAATGGTAATTC #3 score 93.625, 4 gaps 5058 GATTGT---A-GGGAATTC ***.* * **.***** 1 CATTTTCCAATGGTAATTC #4 score 93.625, 4 gaps 5008 AATTTT-C---TTTAATTC ***** * ..****** 1 CATTTTCCAATGGTAATTC #5 score 91.000, 3 gaps 8876 TTTTTTCCCATCGATCTAATTC IT CHAR ****** ** * . ****** 1 CATTTTCCAAT-G-G-TAATTC #6 score 90.000, 5 gaps 1156 CCTCGTTACAATCAAGAGTAAGTC * * .** **** * ****.** 1 CAT-TTTCCAAT---G-GTAATTC #7 score 89.875, 3 gaps 1493 C-TTGCCCATTTATCTAATTC * **. *** *. . ****** 1 CATTTTCCAATG-G-TAATTC #8 score 89.250, 4 gaps 6519 C-TGGT---GTGGTAAGTC * *..* ******.** 1 CATTTTCCAATGGTAATTC #9 score 87.500, 3 gaps 1577 C-TTGTGTAATTGTTAATTTC * **.* ** **.*** *** 1 CATTTTCCAA-TGGTAA-TTC #10 score 87.375, 4 gaps 6575 CTATGCTGCCCTATTTCTAAGTC * **. *. ** **.. ***.** 1 C-ATT-TT-CCAATGG-TAATTC #11 score 87.000, 5 gaps 2724 GAGTTGATACTACTGGCCTAATTC * **. * * * *** ****** 1 CA-TTT-T-CCAATGG--TAATTC If you want to continue press 'y' 10 scores above threshold, 11 unique paths printed Another PRIMER with sequence 'HXB2-INV' - press 'y' If you want to INVERT the LARGE sequence - press 'i' i Sequence 'HXB2-INV' has been inverted PRIMER sequence: Enter name of file containing query sequence, or for 3_primers -> Locus names in the file <3_primers>: sbc4 sbc5 sbc6 Enter locus name of query sequence, or for sbc4 -> 19 bases found for locus sbc4 in file 3_primers Locus "HXB2-INV_INVERTED" (len = 9719) against "sbc4" (len = 19) Position weighting matrix: Default is Val=1 from 5' end to position (length-8) linearly increasing to Val=10 at the 3' end of the primer. If you want to CHANGE it enter 'y' Output file: test File exists: Overwrite, Append, Reenter, or Backup, or for Append -> 2372 maxima #1 score 100.875, 0 gaps 7365 AATTTTTCTACTGTAATTC ***** * * .******* 1 CATTTTCCAATGGTAATTC #2 score 93.500, 3 gaps 9082 GACTGG--AAGGGCTAATTC * *.. **.** ****** 1 CATTTTCCAATGG-TAATTC #3 score 92.500, 5 gaps 4637 CA--GG--AATTTGGAATTC ** .. ** *.*.***** 1 CATTTTCCAA-TGGTAATTC #4 score 89.625, 3 gaps 8043 C-TGTGCC--TTGGAATGC * *.*.** *.*.***.* 1 CATTTTCCAATGGTAATTC #5 score 89.500, 3 gaps 1 ---TGG--AAGGGCTAATTC *.. **.** ****** 1 CATTTTCCAATGG-TAATTC #6 score 83.625, 4 gaps 8343 GAGTTAGGC-A-GGGATATTC *.** . * * **.* **** 1 CATTT-TCCAATGGTA-ATTC #7 score 81.375, 6 gaps 2235 C-TGTATCC---TTTAACTTC * *.* *** ..*** *** 1 CATTT-TCCAATGGTAA-TTC #8 score 80.500, 4 gaps 5347 CA--GACCAA--CTAATTC ** . **** ****** 1 CATTTTCCAATGGTAATTC #9 score 80.375, 6 gaps 2801 CAAGACTT-C--TGGGAAGTTC * *. ** * ***.** *** 1 C-AT-TTTCCAATGGTAA-TTC #10 score 80.000, 4 gaps 2099 CTGGCCTTCCTACAAGGGAAGGC * .. **** * * **.**..* 1 CATT--TTCC-A-ATGGTAATTC #11 score 79.125, 4 gaps 3335 AAATTG--AATTGGGCAAGTC * **. ** ***. **.** 1 CATTTTCCAA-TGGT-AATTC If you want to continue press 'y' 10 scores above threshold, 11 unique paths printed Another PRIMER with sequence 'HXB2-INV_INVERTED' - press 'y' If you want to INVERT the LARGE sequence - press 'i' -------------- end of sample run ----------------------------