eprimer3.txt

emboss的linux版本的源代码

                                  (Any numeric value)
*  -maxdifftm          float      [100.0] Maximum acceptable (unsigned)
                                  difference between the melting temperatures
                                  of the forward and reverse primers. (Any
                                  numeric value)
*  -ogcpercent         float      [50.0] Primer optimum GC percent. (Any
                                  numeric value)
*  -mingc              float      [20.0] Minimum allowable percentage of Gs
                                  and Cs in any primer. (Any numeric value)
*  -maxgc              float      [80.0] Maximum allowable percentage of Gs
                                  and Cs in any primer generated by Primer.
                                  (Any numeric value)
*  -saltconc           float      [50.0] The millimolar concentration of salt
                                  (usually KCl) in the PCR. EPrimer3 uses this
                                  argument to calculate oligo melting
                                  temperatures. (Any numeric value)
*  -dnaconc            float      [50.0] The nanomolar concentration of
                                  annealing oligos in the PCR. EPrimer3 uses
                                  this argument to calculate oligo melting
                                  temperatures. The default (50nM) works well
                                  with the standard protocol used at the
                                  Whitehead/MIT Center for Genome
                                  Research--0.5 microliters of 20 micromolar
                                  concentration for each primer oligo in a 20
                                  microliter reaction with 10 nanograms
                                  template, 0.025 units/microliter Taq
                                  polymerase in 0.1 mM each dNTP, 1.5mM MgCl2,
                                  50mM KCl, 10mM Tris-HCL (pH 9.3) using 35
                                  cycles with an annealing temperature of 56
                                  degrees Celsius. This parameter corresponds
                                  to 'c' in Rychlik, Spencer and Rhoads'
                                  equation (ii) (Nucleic Acids Research, vol
                                  18, num 12) where a suitable value (for a
                                  lower initial concentration of template) is
                                  'empirically determined'. The value of this
                                  parameter is less than the actual
                                  concentration of oligos in the reaction
                                  because it is the concentration of annealing
                                  oligos, which in turn depends on the amount
                                  of template (including PCR product) in a
                                  given cycle. This concentration increases a
                                  great deal during a PCR; fortunately PCR
                                  seems quite robust for a variety of oligo
                                  melting temperatures.
                                  See ADVICE FOR PICKING PRIMERS. (Any numeric
                                  value)
*  -maxpolyx           integer    [5] The maximum allowable length of a
                                  mononucleotide repeat in a primer, for
                                  example AAAAAA. (Integer 0 or more)
*  -productosize       integer    [200] The optimum size for the PCR product.
                                  0 indicates that there is no optimum product
                                  size. (Integer 0 or more)
*  -productsizerange   range      [100-300] The associated values specify the
                                  lengths of the product that the user wants
                                  the primers to create, and is a space
                                  separated list of elements of the form
                                  (x)-(y)
                                  where an (x)-(y) pair is a legal range of
                                  lengths for the product. For example, if one
                                  wants PCR products to be between 100 to 150
                                  bases (inclusive) then one would set this
                                  parameter to 100-150. If one desires PCR
                                  products in either the range from 100 to 150
                                  bases or in the range from 200 to 250 bases
                                  then one would set this parameter to
                                  100-150 200-250.
                                  EPrimer3 favors ranges to the left side of
                                  the parameter string. EPrimer3 will return
                                  legal primers pairs in the first range
                                  regardless the value of the objective
                                  function for these pairs. Only if there are
                                  an insufficient number of primers in the
                                  first range will EPrimer3 return primers in
                                  a subsequent range.
*  -productotm         float      [0.0] The optimum melting temperature for
                                  the PCR product. 0 indicates that there is
                                  no optimum temperature. (Any numeric value)
*  -productmintm       float      [-1000000.0] The minimum allowed melting
                                  temperature of the amplicon. Please see the
                                  documentation on the maximum melting
                                  temperature of the product for details. (Any
                                  numeric value)
*  -productmaxtm       float      [1000000.0] The maximum allowed melting
                                  temperature of the amplicon. Product Tm is
                                  calculated using the formula from Bolton and
                                  McCarthy, PNAS 84:1390 (1962) as presented
                                  in Sambrook, Fritsch and Maniatis, Molecular
                                  Cloning, p 11.46 (1989, CSHL Press).
                                  Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) -
                                  600/length
                                  Where [Na+} is the molar sodium
                                  concentration, (%GC) is the percent of Gs
                                  and Cs in the sequence, and length is the
                                  length of the sequence.
                                  A similar formula is used by the prime
                                  primer selection program in GCG
                                  http://www.gcg.com), which instead uses
                                  675.0/length in the last term (after F.
                                  Baldino, Jr, M.-F. Chesselet, and M.E.
                                  Lewis, Methods in Enzymology 168:766 (1989)
                                  eqn (1) on page 766 without the mismatch and
                                  formamide terms). The formulas here and in
                                  Baldino et al. assume Na+ rather than K+.
                                  According to J.G. Wetmur, Critical Reviews
                                  in BioChem. and Mol. Bio. 26:227 (1991) 50
                                  mM K+ should be equivalent in these formulae
                                  to .2 M Na+. EPrimer3 uses the same salt
                                  concentration value for calculating both the
                                  primer melting temperature and the oligo
                                  melting temperature. If you are planning to
                                  use the PCR product for hybridization later
                                  this behavior will not give you the Tm under
                                  hybridization conditions. (Any numeric
                                  value)
*  -oligoexcludedregion range      [(full sequence)] Middle oligos may not
                                  overlap any region specified by this tag.
                                  The associated value must be a
                                  space-separated list of
                                  (start),(end)
                                  pairs, where (start) is the index of the
                                  first base of an excluded region, and (end)
                                  is the last. Often one would make Target
                                  regions excluded regions for internal
                                  oligos.
*  -oligoinput         string     The sequence of an internal oligo to check
                                  and around which to design forward and
                                  reverse primers. Must be a substring of
                                  SEQUENCE. (Any string is accepted)
*  -oligoosize         integer    [20] Optimum length (in bases) of an
                                  internal oligo. EPrimer3 will attempt to
                                  pick primers close to this length. (Integer
                                  0 or more)
*  -oligominsize       integer    [18] Minimum acceptable length of an
                                  internal oligo. Must be greater than 0 and
                                  less than or equal to
                                  INTERNAL-OLIGO-MAX-SIZE. (Integer 0 or more)
*  -oligomaxsize       integer    [27] Maximum acceptable length (in bases) of
                                  an internal oligo. Currently this parameter
                                  cannot be larger than 35. This limit is
                                  governed by maximum oligo size for which
                                  EPrimer3's melting-temperature is valid.
                                  (Integer up to 35)
*  -oligootm           float      [60.0] Optimum melting temperature (Celsius)
                                  for an internal oligo. EPrimer3 will try to
                                  pick oligos with melting temperatures that
                                  are close to this temperature. The oligo
                                  melting temperature formula in EPrimer3 is
                                  that given in Rychlik, Spencer and Rhoads,
                                  Nucleic Acids Research, vol 18, num 12, pp
                                  6409-6412 and Breslauer, Frank, Bloeker and
                                  Marky, Proc. Natl. Acad. Sci. USA, vol 83,
                                  pp 3746-3750. Please refer to the former
                                  paper for background discussion. (Any
                                  numeric value)
*  -oligomintm         float      [57.0] Minimum acceptable melting
                                  temperature(Celsius) for an internal oligo.
                                  (Any numeric value)
*  -oligomaxtm         float      [63.0] Maximum acceptable melting
                                  temperature (Celsius) for an internal oligo.
                                  (Any numeric value)
*  -oligoogcpercent    float      [50.0] Internal oligo optimum GC percent.
                                  (Any numeric value)
*  -oligomingc         float      [20.0] Minimum allowable percentage of Gs
                                  and Cs in an internal oligo. (Any numeric
                                  value)
*  -oligomaxgc         float      [80.0] Maximum allowable percentage of Gs
                                  and Cs in any internal oligo generated by
                                  Primer. (Any numeric value)
*  -oligosaltconc      float      [50.0] The millimolar concentration of salt
                                  (usually KCl) in the hybridization. EPrimer3
                                  uses this argument to calculate internal
                                  oligo melting temperatures. (Any numeric
                                  value)
*  -oligodnaconc       float      [50.0] The nanomolar concentration of
                                  annealing internal oligo in the
                                  hybridization. (Any numeric value)
*  -oligoselfany       float      [12.00] The maximum allowable local
                                  alignment score when testing an internal
                                  oligo for (local) self-complementarity.
                                  Local self-complementarity is taken to
                                  predict the tendency of oligos to anneal to
                                  themselves The scoring system gives 1.00 for
                                  complementary bases, -0.25 for a match of
                                  any base (or N) with an N, -1.00 for a
                                  mismatch, and -2.00 for a gap. Only
                                  single-base-pair gaps are allowed. For
                                  example, the alignment
                                  5' ATCGNA 3'
                                  || | |
                                  3' TA-CGT 5'
                                  is allowed (and yields a score of 1.75), but
                                  the alignment
                                  5' ATCCGNA 3'
                                  || | |
                                  3' TA--CGT 5'
                                  is not considered. Scores are non-negative,
                                  and a score of 0.00 indicates that there is
                                  no reasonable local alignment between two
                                  oligos. (Number up to 9999.990)
*  -oligoselfend       float      [12.00] The maximum allowable 3'-anchored
                                  global alignment score when testing a single
                                  oligo for self-complementarity.
                                  The scoring system is as for the Maximum
                                  Complementarity argument. In the examples
                                  above the scores are 7.00 and 6.00
                                  respectively. Scores are non-negative, and a
                                  score of 0.00 indicates that there is no
                                  reasonable 3'-anchored global alignment
                                  between two oligos. In order to estimate
                                  3'-anchored global alignments for candidate
                                  oligos, Primer assumes that the sequence
                                  from which to choose oligos is presented 5'
                                  to 3'.
                                  INTERNAL-OLIGO-SELF-END is meaningless when
                                  applied to internal oligos used for
                                  hybridization-based detection, since
                                  primer-dimer will not occur. We recommend
                                  that INTERNAL-OLIGO-SELF-END be set at least
                                  as high as INTERNAL-OLIGO-SELF-ANY. (Number
                                  up to 9999.990)
*  -oligomaxpolyx      integer    [5] The maximum allowable length of an
                                  internal oligo mononucleotide repeat, for
                                  example AAAAAA. (Integer 0 or more)
*  -oligomaxmishyb     float      [12.0] Similar to MAX-MISPRIMING except that
                                  this parameter applies to the similarity of
                                  candidate internal oligos to the library
                                  specified in INTERNAL-OLIGO-MISHYB-LIBRARY.
                                  (Number up to 9999.990)

   Advanced (Unprompted) qualifiers:
   -mispriminglibraryfile infile     The name of a file containing a nucleotide
                                  sequence library of sequences to avoid
                                  amplifying (for example repetitive
                                  sequences, or possibly the sequences of
                                  genes in a gene family that should not be
                                  amplified.) The file must be in (a slightly
                                  restricted) FASTA format (W. B. Pearson and
                                  D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]);
                                  we briefly discuss the organization of this
                                  file below. If this parameter is specified
                                  then EPrimer3 locally aligns each candidate
                                  primer against each library sequence and
                                  rejects those primers for which the local
                                  alignment score times a specified weight
                                  (see below) exceeds MAX-MISPRIMING. (The
                                  maximum value of the weight is arbitrarily
                                  set to 100.0.)