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NAME

       melting - nearest-neighbor computation of nucleic acid hybridation

SYNOPSIS

       melting [options]

DESCRIPTION

       Melting  computes,  for  a  nucleic  acid  duplex, the enthalpy and the
       entropy of the helix-coil transition, and then its melting temperature.
       Three  types  of  hybridisation  are  possible:  DNA/DNA,  DNA/RNA, and
       RNA/RNA.  The program uses the method of nearest-neighbors. The set  of
       thermodynamic  parameters can be easely changed, for instance following
       an experimental breakthrough. Melting is a free program in  both  sense
       of  the  term. It comes with no cost and it is open-source. In addition
       it is coded in ISO C and can be compiled on any operating system.  Some
       perl scripts are provided to show how melting can be used as a block to
       construct more ambitious programs.

OPTIONS

       The options are treated sequentially. If there is  a  conflict  between
       the value of two options, the latter normally erases the former.

       -Afile.nn
              Informs  the  program  to  use  file.nn as an alternative set of
              nearest-neighbor parameters, rather than  the  default  for  the
              specified  hybridisation  type.  The  standard  distribution  of
              melting provides some files ready-to-use: all97a.nn  (Allawi  et
              al   1997),   bre86a.nn   (Breslauer   et  al  1986),  san96a.nn
              (SantaLucia  et  al  1996),  sug96a.nn  (Sugimoto  et  al  1996)
              san04a.nn  (Santalucia  et al 2004) (DNA/DNA), fre86a.nn (Freier
              et  al  1986),  xia98a.nn  (Xia  et  al  1998),  (RNA/RNA),  and
              sug95a.nn (Sugimoto et al 1995), (DNA/RNA).

              The  program  will  look  for  the file in a directory specified
              during the installation. However,  if  an  environment  variable
              NN_PATH  is  defined,  melting will search in this one first. Be
              careful, the option -A changes the default parameter set defined
              by the option -H.

       -Ccomplementary_sequence
              Enters the complementary sequence, from 3’ to 5’. This option is
              mandatory if there are mismatches between the two strands. If it
              is  not  used,  the program will compute it as the complement of
              the sequence entered with the option -S.

       -Ddnadnade.nn
              Informs the program to use the file dnadnade.nn to  compute  the
              contribution of dangling ends to the thermodynamic of helix-coil
              transition. The dangling ends are not taken into account by  the
              approximative mode.

       -Ffactor
              This  is  the a correction factor used to modulate the effect of
              the  nucleic  acid  concentration  in  the  computation  of  the
              melting temperature. See section ALGORITHM for details.

       -Gx.xxe-xx
              Magnesium   concentration   (No  maximum  concentration  for the
              moment). The effect
                 of  ions  on   thermodynamic   stability   of  nucleic   acid
              duplexes is complex,
                 and   the   correct­ing   functions   are    at   best  rough
              approximations.The published
                 Tm  correction formula for divalent Mg2+  ions  of   Owczarzy
              et al(2008) can
                 take  in  account  the  competitive binding of monovalent and
              divalent ions on DNA.
                 However this formula is only for DNA duplexes.

       -h     Displays a short help and quit with EXIT_SUCCESS.

       -Hhybridisation_type
              Specifies the hybridisation type. This  will  set  the  nearest-
              neighbor  set  to  use  if no alternative set is provided by the
              option -A (remember the options are read sequentially). Moreover
              this  parameter  determines  the equation to use if the sequence
              length exceeds the limit of application of the  nearest-neighbor
              approach (arbitrarily set up by the author). Possible values are
              dnadna, dnarna and rnadna (synonymous), and rnarna.  For reasons
              of  compatibility the values of the previous versions of melting
              A,B,C,F,R,S,T,U,W  are   still   available   although   strongly
              deprecated.  Use  the option -A to require an alternative set of
              thermodynamic parameters. IMPORTANT: If the duplex is a  DNA/RNA
              heteroduplex,  the  sequence of the DNA strand has to be entered
              with the option -S.

       -Iinput_file
              Provides the name of an input file containing the parameters  of
              the  run.  The  input  has  to  contain  one parameter per line,
              formatted as in the command line. The order is not important, as
              well as blank lines. example:

              ###beginning###
              -Hdnadna
              -Asug96a.nn
              -SAGCTCGACTC
              -CTCGAGGTGAG
              -N0.2
              -P0.0001
              -v
              -Ksan96a

              ###end###

       -ifile.nn
                Informs  the  program to use file.nn as an alternative set  of
              inosine pair
                parameters, rather  than   the   default   for  the  specified
              hybridisation type.
                The  standard  distribution  of  melting  provides some  files
              ready-to-use:  san05a.nn
               (Santalucia et al  2005)  for  deoxyinosine  in  DNA  duplexes,
              bre07a.nn (Brent M Znosko
                et  al  2007)for  inosine in RNA duplexes. Note  that  not all
              the inosine mismatched
                wobble’s pairs have been investigated. Therefore it  could  be
              impossible to  compute
                the Tm of a duplex with inosine pairs. Moreover, those inosine
              pairs are not taken
                into account by the  approximative mode.

       -Ksalt_correction
              Permits to chose another correction  for  the  concentration  in
              sodium. Currently, one can chose between wet91a, san96a, san98a.
              See section ALGORITHM.  TP.  BI. "-k" "x.xxe-xx"
                 Potassium  concentration  (No maximum concentration  for  the
              moment). The effect of ions
                 on   thermodynamic   stability   of nucleic  acid duplexes is
              complex, and the correct­ing
                 functions are  at  best rough   approximations.The  published
              Tm  correction formula for
                 sodium  ions  of  Owczarzy  et  al  (2008)is  therefore  also
              applicable to buffers containing Tris or
                 KCl. Monovalent K+, Na+, Tris+ ions  stabilize  DNA duplexes
                 with similar potency, and their effects on  duplex  stability
              are additive. However this formula
                 is only for DNA duplexes.

       -L     Prints the legal informations and quit with EXIT_SUCCESS.

       -Mdnadnamm.nn
              Informs  the  program to use the file dnadnamm.nn to compute the
              contribution of mismatches to the  thermodynamic  of  helix-coil
              transition.  Note that not all the mismatched Crick’s pairs have
              been investigated. Therefore it could be impossible  to  compute
              the  Tm  of  a mismatched duplex. Moreover, those mismatches are
              not taken into account by the approximative mode.

       -Nx.xxe-xx
              Sodium concentration (between 0 and 10 M). The effect of ions on
              thermodynamic
                stability  of  nucleic  acid  duplexes  is  complex,  and  the
              correcting functions
                are at best rough approximations. Moreover, they are generally
              reliable only
                for  [Na+]  belonging to [0.1,10M]. If there are no other ions
              in
                solution, we can use only the sodium correction. In the  other
              case, we use the Owczarzy’s
                algorithm.

       -Ooutput_file
              The  output  is  directed  to  this file instead of the standard
              output. The name of the file can be omitted. An  automatic  name
              is  then  generated, of the form meltingYYYYMMMDD_HHhMMm.out (of
              course, on POSIX compliant systems, you can  emulate  this  with
              the redirection of stdout to a file constructed with the program
              date).

       -Px.xxe-xx
              Concentration of the nucleic acid strand in  excess  (between  0
              and 0.1 M).

       -p     Return   the   directory   supposed   to  contain  the  sets  of
              calorimetric parameters  and  quit  with  EXIT_SUCCESS.  If  the
              environment  variable NN_PATH is set, it is returned. Otherwise,
              the value defined by default during the compilation is returned.

       -q     Turn   off   the   interactive  correction  of  wrongly  entered
              parameter. Useful for run through a server, or a  batch  script.
              Default  is  OFF (i.e. interactive on). The switch works in both
              sens.  Therefore if -q has been set in an input file, another -q
              on  the  command line will switch the quiet mode OFF (same thing
              if two -q are set on the same command line).

       -Ssequence
              Sequence of one strand of the nucleic acid duplex, entered 5’ to
              3’.  IMPORTANT: If it is a DNA/RNA heteroduplex, the sequence of
              the DNA strand has to be  entered.  Uridine  and  thymidine  are
              considered as identical. The bases can be upper or lowercase.

       -Txxx  Size  threshold  before  approximative computation. The nearest-
              neighbour approach will be  used  only  if  the  length  of  the
              sequence is inferior to this threshold.

       -tx.xxe-xx
              Tris  buffer   concentration   (No maximum concentration for the
              moment).
                 The  effect   of   ions   on   thermodynamic   stability   of
              nucleic  acid
                 duplexes  is  complex,  and the correct­ing functions are  at
              best
                 rough  approximations.The published  Tm   correction  formula
              for sodium ions of
                 Owczarzy  et  al(2008)is therefore also applicable to buffers
              containing Tris or
                 KCl. Monovalent K+, Na+, Tris+ ions  stabilize  DNA  duplexes
              with similar potency, and
                 their  effects on duplex stability are additive. However this
              formula is only for DNA
                 duplexes. Be careful, the Tris+ ion  concentration  is  about
              half of the total tris buffer
                 concentration.

       -v     Control  the  verbose mode, issuing a lot more information about
              the current run (try it once to see if  you  can  get  something
              interesting).  Default  is  OFF.  The switch works in both sens.
              Therefore if -v has been set in an input file, another -v on the
              command line will switch the verbose mode OFF (same thing if two
              -v are set on the same command line).

       -V     Displays the version number and quit with EXIT_SUCCESS.

       -x     Force the program to compute an approximative tm, based  on  G+C
              content. This option has to be used with caution. Note that such
              a calcul is increasingly incorrect when the length of the duplex
              decreases.  Moreover, it does not take into account nucleic acid
              concentration, which is a strong mistake.

ALGORITHM

   Thermodynamics of helix-coil transition of nucleic acid
       The nearest-neighbor approach is based on the fact that the  helix-coil
       transition  works  as  a  zipper.   After  an  initial  attachment, the
       hybridisation propagates laterally.  Therefore, the process depends  on
       the  adjacent  nucleotides  on  each  strand  (the Crick’s pairs).  Two
       duplexes with the same base pairs could have different stabilities, and
       on  the  contrary,  two duplexes with different sequences but identical
       sets of Crick’s pairs will have the same thermodynamics properties (see
       Sugimoto  et  al. 1994).  This program first computes the hybridisation
       enthalpy and entropy from the elementary  parameters  of  each  Crick’s
       pair.

       DeltaH = deltaH(initiation) + SUM(deltaH(Crick’s pair))
       DeltaS = deltaS(initiation) + SUM(deltaS(Crick’s pair))

       See  Wetmur  J.G.  (1991) and SantaLucia (1998) for deep reviews on the
       nucleic acid hybridisation and on the different set of nearest-neighbor
       parameters.

   Effect of mismatches and dangling ends
       The  mismatching  pairs  are  also  taken  into  account.  However  the
       thermodynamic parameters are still not  available  for  every  possible
       cases (notably when both positions are mismatched). In such a case, the
       program, unable to compute  any  relevant  result,  will  quit  with  a
       warning.

       The  two  first and positions cannot be mismatched. in such a case, the
       result is unpredictable, and all cases are possible. for instance  (see
       Allawi and SanLucia 1997), the duplex

       A          T
        GTGAGCTCAT
        TACTCGAGTG
       T          A

       is more stable than

       AGTGAGCTCATT
       TTACTCGAGTGA

       The  dangling  ends,  that is the umatched terminal nucleotides, can be
       taken into account.

   Example
       DeltaH(
       AGCGATGAA-
       -CGCTGCTTT
       ) = DeltaH(AG/-C)+DeltaH(A-/TT)
       +DeltaH(initG/C)+DeltaH(initA/T)
       +DeltaH(GC/CG)+DeltaH(CG/GC)+2xDeltaH(GA/CT)+DeltaH(AA/TT)
       +Delta(AT/TG mismatch) +DeltaG(TC/GG mismatch)

       (The same computation is performed for DeltaS)

   The melting temperature
       Then the melting temperature is computed by the following formula:

       Tm = DeltaH / (DeltaS + Rx ln ([nucleic acid]/F))
       Tm in K (for [Na+] = 1 M )
            + f([Na+]) - 273.15
       correction for the salt concentration (if there are only sodium cations
       in the solution)and to get the temperature in degree Celsius.  (In fact
       some corrections are directly  included  in  the  DeltaS  see  that  of
       SanLucia 1998)

   Correction for the concentration of nucleic acid
       If  the concentration of the two strands are similar, F is 1 in case of
       self-complementary oligonucleotides, 4 otherwise. If one strand  is  in
       excess  (for  instance in PCR experiment), F is 2 (Actually the formula
       would have to use the difference  of  concentrations  rather  than  the
       total  concentration,  but  if  the  excess  is  sufficient,  the total
       concentration can be assumed to be identical to  the  concentration  of
       the strand in excess).

       Note  however,  MELTING  makes the assumption of no self-assembly, i.e.
       the computation does not take any entropic term to  correct  for  self-
       complementarity.

   Correction for the concentration of salt
       If there are only sodium ions in the solution, we can use the following
       corrections:

       The correction can be chosen between wet91a, presented in  Wetmur  1991
       i.e.
       16.6 x log([Na+] / (1 + 0.7 x [Na+])) + 3.85

       san96a presented in SantaLucia et al. 1996 i.e.
       12.5 x log[Na+]

       and  san98a  presented  in  SantaLucia  1998  i.e.  a correction of the
       entropic term without modification of enthalpy
       DeltaS = DeltaS([Na+]=1M) + 0.368 x (N-1) x ln[Na+]

       Where N is the length of the duplex (SantaLucia 1998 actually used  ’N’
       the number of non-terminal phosphates, that is effectively equal to our
       N-1). CAUTION, this correction  is  meant  to  correct  entropy  values
       expressed in cal.mol-1.K-1!!!

   Correction for the concentration of ions when other monovalent ions such as
       Tris+ and K+ or divalent Mg2+ ions are added
       If there are  only  Na+  ions,  we  can  use  the  correction  for  the
       concentration  of  salt(see  above). In the opposite case , we will use
       the magnesium and  monovalent  ions  correction  from  Owczarzy  et  al
       (2008). (only for DNA duplexes)

       [Mon+] = [Na+] + [K+] + [Tris+]

       Where  [Tris+]  =  [Tris  buffer]/2.  (in the option -t, it is the Tris
       buffer concentration which is entered).

       If [Mon+] = 0, the divalent ions are the only ions present
        and the melting temperature is :

       1/Tm(Mg2+) = 1/Tm(1M Na+) + a - b x  ln([Mg2+])  +  Fgc  x  (c  +  d  x
       ln([Mg2+])  + 1/(2 x (Nbp - 1)) x (- e +f x ln([Mg2+]) + g x ln([Mg2+])
       x ln([Mg2+]))

       where : a = 3.92/100000.  b = 9.11/1000000.   c  =  6.26/100000.   d  =
       1.42/100000.   e  = 4.82/10000.  f = 5.25/10000.  g = 8.31/100000.  Fgc
       is the fraction of GC base pairs in the sequence and Nbp is the  length
       of the sequence (Number of base pairs).

       If  [Mon+]  >  0,  there  are  several  cases  because  we  can  have a
       competitive DNA binding between monovalent and divalent cations  :

       If the ratio [Mg2+]^(0.5)/[Mon+] is inferior to  0.22,  monovalent  ion
       influence  is  dominant,  divalent  cations  can be disregarded and the
       melting temperature is :

       1/Tm(Mg2+) = 1/Tm(1M Na+) + (4.29 x Fgc - 3.95) x 1/100000 x ln([mon+])
       + 9.40 x 1/1000000 x ln([Mon+]) x ln([Mon+])

       where : Fgc is the fraction of GC base pairs in the sequence.

       If the ratio [Mg2+]^(0.5)/[Mon+] is included in [0.22, 6[, we must take
       in account both Mg2+ and monovalent cations concentrations. The melting
       temperature is :

       1/Tm(Mg2+)  =  1/Tm(1M  Na+)  +  a  -  b  x ln([Mg2+]) + Fgc x (c + d x
       ln([Mg2+]) + 1/(2 x (Nbp - 1)) x (- e + f x ln([Mg2+]) + g x ln([Mg2+])
       x ln([Mg2+]))

       where : a = 3.92/100000 x (0.843 - 0.352 x [Mon+]0.5 x ln([Mon+])).
               b = 9.11/1000000.       c = 6.26/100000.
               d = 1.42/100000 x (1.279 - 4.03/1000 x ln([mon+]) - 8.03/1000 x
            ln([mon+]  x  ln([mon+]).        e   =   4.82/10000.         f   =
       5.25/10000.        g  =  8.31/100000  x  (0.486  - 0.258 x ln([mon+]) +
       5.25/1000 x ln([mon+] x ln([mon+] x ln([mon+]).

       Fgc is the fraction of GC base pairs in the sequence  and  Nbp  is  the
       length of the sequence (Number of base pairs).

       Finally,  if  the  ratio [Mg2+]^(0.5)/[Mon+] is superior to 6, divalent
       ion influence is dominant, monovalent cations can  be  disregarded  and
       the melting temperature is :

       1/Tm(Mg2+)  =  1/Tm(1M  Na+)  +  a  -  b  x ln([Mg2+]) + Fgc x (c + d x
       ln([Mg2+]) + 1/(2 x (Nbp - 1)) x (- e + f x ln([Mg2+]) + g x ln([Mg2+])
       x ln([Mg2+]))

       where  :  a  =  3.92/100000.   b = 9.11/1000000.  c = 6.26/100000.  d =
       1.42/100000.  e = 4.82/10000.  f = 5.25/10000.  g = 8.31/100000.

       Fgc is the fraction of GC base pairs in the sequence  and  Nbp  is  the
       length of the sequence (Number of base pairs).

   Long sequences
       It  is important to realise that the nearest-neighbor approach has been
       established on small oligonucleotides. Therefore the use of melting  in
       the non-approximative mode is really accurate only for relatively short
       sequences (Although if the sequences are two short, let’s say <  6  bp,
       the  influence of extremities becomes too important and the reliability
       decreases a lot). For long sequences an  approximative  mode  has  been
       designed.  This  mode is launched if the sequence length is higher than
       the value given by the option -T (the default threshold is 60 bp).

       The melting temperature is computed by the following formulas:

       DNA/DNA:
       Tm = 81.5+16.6*log10([Na+]/(1+0.7[Na+]))+0.41%GC-500/size

       DNA/RNA:
       Tm = 67+16.6*log10([Na+]/(1.0+0.7[Na+]))+0.8%GC-500/size

       RNA/RNA:
       Tm = 78+16.6*log10([Na+]/(1.0+0.7[Na+]))+0.7%GC-500/size

       This mode is nevertheless strongly disencouraged.

   Miscellaneous comments
       Melting is currently accurate only when the hybridisation is  performed
       at pH 71.

       The  computation  is  valid  only  for  the hybridisations performed in
       aqueous medium.   Therefore  the  use  of  denaturing  agents  such  as
       formamide completely invalidates the results.

REFERENCES

       Allawi  H.T., SantaLucia J. (1997).  Thermodynamics and NMR of internal
       G.T mismatches in DNA.  Biochemistry 36: 10581-10594

       Allawi H.T., SantaLucia J.  (1998).   Nearest  Neighbor  thermodynamics
       parameters  for  internal  G.A  mismatches  in  DNA.   Biochemistry 37:
       2170-2179

       Allawi H.T., SantaLucia J.  (1998).   Thermodynamics  of  internal  C.T
       mismatches in DNA.  Nucleic Acids Res 26: 2694-2701.

       Allawi  H.T., SantaLucia J. (1998).  Nearest Neighbor thermodynamics of
       internal A.C mismatches in DNA: sequence  dependence  and  pH  effects.
       Biochemistry 37: 9435-9444.

       Bommarito   S.,   Peyret   N.,  SantaLucia  J.  (2000).   Thermodynamic
       parameters for DNA sequences with dangling ends.  Nucleic Acids Res 28:
       1929-1934

       Breslauer  K.J.,  Frank R., Bl�ker H., Marky L.A. (1986).  Predicting
       DNA duplex stability from the base sequence.  Proc Natl  Acad  Sci  USA
       83: 3746-3750

       Freier  S.M.,  Kierzek  R.,  Jaeger  J.A., Sugimoto N., Caruthers M.H.,
       Neilson T., Turner D.H. (1986).  Improved  free-energy  parameters  for
       predictions of RNA duplex stability.  Biochemistry 83:9373-9377

       Owczarzy  R.,  Moreira  B.G.,  You Y., Behlke M.B., Walder J.A.  (2008)
       Predicting stability of DNA duplexes in solutions containing  Magnesium
       and Monovalent Cations. Biochemistry 47: 5336-5353.

       Peyret  N.,  Seneviratne  P.A.,  Allawi  H.T.,  SantaLucia  J.  (1999).
       Nearest Neighbor thermodynamics and NMR of DNA sequences with  internal
       A.A,   C.C,  G.G  and  T.T  mismatches.   dependence  and  pH  effects.
       Biochemistry 38: 3468-3477

       SantaLucia J. Jr,  Allawi  H.T.,  Seneviratne  P.A.  (1996).   Improved
       nearest-neighbor   parameters  for  predicting  DNA  duplex  stability.
       Biochemistry 35: 3555-3562

       Sugimoto N., Katoh  M.,  Nakano  S.,  Ohmichi  T.,  Sasaki  M.  (1994).
       RNA/DNA  hybrid duplexes with identical nearest-neighbor base-pairs hve
       identical stability.  FEBS Letters 354: 74-78

       Sugimoto N., Nakano S., Katoh M., Matsumura A.,  Nakamuta  H.,  Ohmichi
       T., Yoneyama M., Sasaki M. (1995).  Thermodynamic parameters to predict
       stability of RNA/DNA hybrid duplexes.  Biochemistry 34: 11211-11216

       Sugimoto N.,  Nakano  S.,  Yoneyama  M.,  Honda  K.  (1996).   Improved
       thermodynamic   parameters  and  helix  initiation  factor  to  predict
       stability of DNA duplexes.  Nuc Acids Res 24: 4501-4505

       Watkins  N.E.,  Santalucia   J.   Jr.   (2005).   Nearest-neighbor   t-
       hermodynamics  of  deoxyinosine  pairs  in  DNA duplexes. Nucleic Acids
       Research 33: 6258-6267

       Wright D.J., Rice J.L.,  Yanker  D.M.,  Znosko  B.M.  (2007).   Nearest
       neighbor   parameters   for  inosine-uridine  pairs  in  RNA  duplexes.
       Biochemistry 46: 4625-4634

       Xia T., SantaLucia J., Burkard M.E., Kierzek R., Schroeder  S.J.,  Jiao
       X.,  Cox  C.,  Turner  D.H.  (1998).   Thermodynamics parameters for an
       expanded nearest-neighbor model for  formation  of  RNA  duplexes  with
       Watson-Crick base pairs.  Biochemistry 37: 14719-14735

       For review see:

       SantaLucia   J.  (1998)  A  unified  view  of  polymer,  dumbbell,  and
       oligonucleotide DNA nearest-neighbor thermodynamics.   Proc  Natl  Acad
       Sci USA 95: 1460-1465

       SantaLucia    J.,   Hicks  Donald  (2004)  The  Thermodynamics  of  DNA
       structural motifs. Annu. Rev. Biophys. Struct. 33: 415 -440

       Wetmur J.G. (1991)  DNA  probes:  applications  of  the  principles  of
       nucleic acid hybridization.  Crit Rev Biochem Mol Biol 26: 227-259

FILES

       *.nn   Files  containing  the nearest-neighbor parameters, enthalpy and
              entropy, for each Crick’s pair.  They have to  be  placed  in  a
              directory  defined  during  the  compilation  or targeted by the
              environment variable NN_PATH.

       tkmelting.pl
              A Graphical User Interface written in Perl/Tk is  available  for
              those who prefer the ’button and menu’ approach.

       *.pl   Scripts  are available to use MELTING iteratively. For instance,
              the script  multi.pl  permits  to  predict  the  Tm  of  several
              duplexes  in one shot. The script profil.pl allow an interactive
              computation along a sequence, by sliding a window  of  specified
              width.

SEE ALSO

       New    versions    and    related    material    can    be   found   at
       http://www.pasteur.fr/recherche/unites/neubiomol/meltinghome.html   and
       at at https://sourceforge.net/projects/melting/

       You     can     use     MELTING     through    a    web    server    at
       http://bioweb.pasteur.fr/seqanal/interfaces/melting.html

KNOWN BUGS

       The infiles have to be ended by a blank line because otherwise the last
       line is not decoded.

       If  an  infile is called, containing the address of another input file,
       it does not care of this latter.  If it is its own address, the program
       quit (is it a bug or a feature?).

       In  interactive mode, a sequence can be entered on several lines with a
       backslash

       AGCGACGAGCTAGCCTA\
       AGGACCTATACGAC

       If by mistake it is entered as

       AGCGACGAGCTAGCCTA\AGGACCTATACGAC

       The backslash will be considered as an illegal character. Here again, I
       do  not  think it is actually a bug (even if it is unlikely, there is a
       small probability that the  backslash  could  actually  be  a  mistyped
       base).

COPYRIGHT

       Melting  is  copyright  (C) 1997, 2009 by Nicolas Le Novère and Marine
       Dumousseau

       This program is free software; you can redistribute it and/or modify it
       under  the  terms of the GNU General Public License as published by the
       Free Software Foundation; either version 2 of the License, or (at  your
       option) any later version.

       This  program  is  distributed  in the hope that it will be useful, but
       WITHOUT  ANY  WARRANTY;  without   even   the   implied   warranty   of
       MERCHANTABILITY  or  FITNESS  FOR  A  PARTICULAR  PURPOSE.  See the GNU
       General Public License for more details.

       You should have received a copy of the GNU General Public License along
       with this program; if not, write to the Free Software Foundation, Inc.,
       59 Temple Place, Suite 330, Boston, MA  02111-1307 USA

ACKNOWLEDGEMENTS

       Nicolas Joly is an efficient and kind debugger and advisor.   Catherine
       Letondal wrote the HTML interface to melting. Thanks to Nirav Merchant,
       Taejoon Kwon, Leo Schalkwyk, Mauro Petrillo, Andrew Thompson, Wong Chee
       Hong,  Ivano  Zara for their bug fixes and comments.  Thanks to Richard
       Owczarzy for his magnesium correction. Thanks to Charles Plessy for the
       graphical  interface  files.   Finally  thanks  to  the usenet helpers,
       particularly Olivier Dehon and Nicolas Chuche.

AUTHORS

       Nicolas Le Novère  and  Marine  Dumousseau,  EMBL-EBI,  Wellcome-Trust
       Genome Campus Hinxton Cambridge, CB10 1SD, UK lenov@ebi.ac.uk

HISTORY

       See  the  file  ChangeLog  for  the  changes of the versions 4 and more
       recent.