primer3.ihelp

emboss的linux版本的源代码

                                  the amplicon. Please see the documentation
                                  on the maximum melting temperature of the
                                  product for details.
*  -productmaxtm       float      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+. Primer3 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.
*  -oligoexcludedregion range      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.
*  -oligoosize         integer    Optimum length (in bases) of an internal
                                  oligo. Primer3 will attempt to pick primers
                                  close to this length.
*  -oligominsize       integer    Minimum acceptable length of an internal
                                  oligo. Must be greater than 0 and less than
                                  or equal to INTERNAL-OLIGO-MAX-SIZE.
*  -oligomaxsize       integer    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
                                  Primer3's melting-temperature is valid.
*  -oligootm           float      Optimum melting temperature (Celsius) for an
                                  internal oligo. Primer3 will try to pick
                                  oligos with melting temperatures that are
                                  close to this temperature. The oligo melting
                                  temperature formula in Primer3 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.
*  -oligomintm         float      Minimum acceptable melting
                                  temperature(Celsius) for an internal oligo.
*  -oligomaxtm         float      Maximum acceptable melting temperature
                                  (Celsius) for an internal oligo.
*  -oligoogcpercent    float      Internal oligo optimum GC percent.
*  -oligomingc         float      Minimum allowable percentage of Gs and Cs in
                                  an internal oligo.
*  -oligomaxgc         float      Maximum allowable percentage of Gs and Cs in
                                  any internal oligo generated by Primer.
*  -oligosaltconc      float      The millimolar concentration of salt
                                  (usually KCl) in the hybridization. Primer3
                                  uses this argument to calculate internal
                                  oligo melting temperatures.
*  -oligodnaconc       float      The nanomolar concentration of annealing
                                  internal oligo in the hybridization.
*  -oligoselfany       float      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.
*  -oligoselfend       float      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.
*  -oligomaxpolyx      integer    The maximum allowable length of an internal
                                  oligo mononucleotide repeat, for example
                                  AAAAAA.

   Advanced qualifiers:
   -explainflag        bool       If this flag is non-0, produce LEFT-EXPLAIN,
                                  RIGHT-EXPLAIN, and INTERNAL-OLIGO-EXPLAIN
                                  output tags, which are intended to provide
                                  information on the number of oligos and
                                  primer pairs that Primer3 examined, and
                                  statistics on the number discarded for
                                  various reasons.
   -fileflag           bool       If the associated value is non-0, then
                                  Primer3 creates two output files for each
                                  input SEQUENCE. File (sequence-id).for lists
                                  all acceptable forward primers for
                                  (sequence-id), and (sequence-id).rev lists
                                  all acceptable reverse primers for
                                  (sequence-id), where (sequence-id) is the
                                  value of the SEQUENCE-ID tag (which must be
                                  supplied). In addition, if the input tag
                                  TASK is 1 or 4, Primer3 produces a file
                                  (sequence-id).int, which lists all
                                  acceptable internal oligos.
   -firstbaseindex     integer    This parameter is the index of the first
                                  base in the input sequence. For input and
                                  output using 1-based indexing (such as that
                                  used in GenBank and to which many users are
                                  accustomed) set this parameter to 1. For
                                  input and output using 0-based indexing set
                                  this parameter to 0. (This parameter also
                                  affects the indexes in the contents of the
                                  files produced when the primer file flag is
                                  set.)
   -pickanyway         bool       If true pick a primer pair even if
                                  LEFT-INPUT, RIGHT-INPUT, or
                                  INTERNAL-OLIGO-INPUT violates specific
                                  constraints.
   -mispriminglibrary  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