Restriction Site Search Programs

Biochemical Engineering

Analysis of pBR322 provides an example on the use the restriction site search programs and the partial digest programs.

Here is another example with the sequence of Photinus pyralis (North American firefly) luciferase gene (firefly.seq).

First, find the peptide sequence and store it in a file. For example, firefly liciferase has the the following DNA sequence, which was simply copied/pasted from the genetic database..

        1 ctgcagaaat aactaggtac taagcccgtt tgtgaaaagt ggccaaaccc ataaatttgg
       61 caattacaat aaagaagcta aaattgtggt caaactcaca aacattttta ttatatacat
      121 tttagtagct gatgcttata aaagcaatat ttaaatcgta aacaacaaat aaaataaaat
      181 ttaaacgatg tgattaagag ccaaaggtcc tctagaaaaa ggtatttaag caacggaatt
      241 cctttgtgtt acattcttga atgtcgctcg cagtgacatt agcattccgg tactgttggt
      301 aaaatggaag acgccaaaaa cataaagaaa ggcccggcgc cattctatcc tctagaggat
      361 ggaaccgctg gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca
      421 attgcttttg tgagtatttc tgtctgattt ctttcgagtt aacgaaatgt tcttatgttt
      481 ctttagacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc
      541 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta
      601 tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt
      661 gcagttgcgc ccgcgaacga catttataat gaacgtaagc accctcgcca tcagaccaaa
      721 gggaatgacg tatttaattt ttaaggtgaa ttgctcaaca gtatgaacat ttcgcagcct
      781 accgtagtgt ttgtttccaa aaaggggttg caaaaaattt tgaacgtgca aaaaaaatta
      841 ccaataatcc agaaaattat tatcatggat tctaaaacgg attaccaggg atttcagtcg
      901 atgtacacgt tcgtcacatc tcatctacct cccggtttta atgaatacga ttttgtacca
      961 gagtcctttg atcgtgacaa aacaattgca ctgataatga attcctctgg atctactggg
     1021 ttacctaagg gtgtggccct tccgcataga actgcctgcg tcagattctc gcatgccagg
     1081 tatgtcgtat aacaagagat taagtaatgt tgctacacac attgtagaga tcctattttt
     1141 ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca tcacggtttt
     1201 ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt aatgtataga
     1261 tttgaagaag agctgttttt acgatccctt caggattaca aaattcaaag tgcgttgcta
     1321 gtaccaaccc tattttcatt cttcgccaaa agcactctga ttgacaaata cgatttatct
     1381 aatttacacg aaattgcttc tgggggcgca cctctttcga aagaagtcgg ggaagcggtt
     1441 gcaaaacggt gagttaagcg cattgctagt atttcaaggc tctaaaacgg cgcgtagctt
     1501 ccatcttcca gggatacgac aaggatatgg gctcactgag actacatcag ctattctgat
     1561 tacacccgag ggggatgata aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc
     1621 gaaggttgtg gatctggata ccgggaaaac gctgggcgtt aatcagagag gcgaattatg
     1681 tgtcagagga cctatgatta tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt
     1741 gattgacaag gatggatggc tacattctgg agacatagct tactgggacg aagacgaaca
     1801 cttcttcata gttgaccgct tgaagtcttt aattaaatac aaaggatatc aggtaatgaa
     1861 gatttttaca tgcacacacg ctacaatacc tgtaggtggc ccccgctgaa ttggaatcga
     1921 tattgttaca acaccccaac atcttcgacg cgggcgtggc aggtcttccc gacgatgacg
     1981 ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg acggaaaaag
     2041 agatcgtgga ttacgtcgcc agtaaatgaa ttcgttttac gttactcgta ctacaattct
     2101 tttcataggt caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga
     2161 agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa
     2221 ggccaagaag ggcggaaagt ccaaattgta aaatgtaact gtattcagcg atgacgaaat
     2281 tcttagctat tgtaatatta tatgcaaatt gatgaatggt aattttgtaa ttgtgggtca
     2341 ctgtactatt ttaacgaata ataaaatcag gtataggtaa ctaaaaa

Place the sequence in a file (firefly.sq1). Run one of the following programs that find all the sites recognized by the restriction enzymes from Sigma. Issue the following command from DOS to run the program. "DOS>" is the DOS prompt, which you don't type; the less-than sign "<" tells the program to receive response from the specified file named "firefly.res", which gives the file containing the gene sequence, the start/end of exons, and the various sequences recognized by the Sigma restriction enzymes. I created the response file by modifying the list of restriction enzymes from Sigma. The file needs to be modified only slightly to analyze other sequences. The second greater-than sign ">" in the command line below is to tell the program to redirect the output to a file rather than to the screen so that we can review the information at our leisure.

    DOS>gene4 <firefly.res >firefly.out

Restriction Site Search Programs

FORTRAN Version

FORTRAN Source Code (gene3.for)
Executable PC Program (gene3.exe)

Quick BASIC Version
- Quick BASIC Source Code (gene3b.bas)
- Executable PC Program (gene3b.exe)
FORTRAN, /w Codon Translation
- FORTRAN Source Code (gene4.for)
- Executable PC Program (gene4.exe)

Above programs also give mRNA sequence from the exon information and translate the mRNA sequence into the corresponding peptide sequence. The following file contains result of running one of the above programs.

Primary mRNA Sequence (firefly.sq2)

Note: There are serveral public domain programs that help with restriction site analysis.

Partial Digest Program

Partial Digest Program
- FORTRAN Source Code (digest.for)
- Executable PC Program (digest.exe)
EcoRII Partial Digest of pBR322
- PDF Version (pbr322.pdf)
- Mathcad File (pbr322.mcd)
- Data on Base Location for the Above File (pbr322.prn)

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Biochemical Engineering -- Restriction Site Search Programs
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Nam Sun Wang

Department of Chemical & Biomolecular Engineering

University of Maryland

College Park, MD 20742-2111

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