primer3.ihelp

emboss的linux版本的源代码

   Mandatory qualifiers:
  [-sequence]          seqall     The sequence from which to choose primers.
                                  The sequence must be presented 5' to 3'
  [-outfile]           outfile    Output file name

   Optional qualifiers (* if not always prompted):
   -task               list       Tell Primer3 what task to perform. Legal
                                  values are 0: 'Pick PCR primers', 1: 'Pick
                                  PCR primers and hybridization probe', 2:
                                  'Pick forward primer only', 3: 'Pick reverse
                                  primer only', 4: 'Pick hybridization probe
                                  only'.
                                  The tasks should be self explanatory.
                                  Briefly, an 'internal oligo' is intended to
                                  be used as a hybridization probe (hyb probe)
                                  to detect the PCR product after
                                  amplification.
   -numreturn          integer    The maximum number of primer pairs to
                                  return. Primer pairs returned are sorted by
                                  their 'quality', in other words by the value
                                  of the objective function (where a lower
                                  number indicates a better primer pair).
                                  Caution: setting this parameter to a large
                                  value will increase running time.
   -includedregion     range      A sub-region of the given sequence in which
                                  to pick primers. For example, often the
                                  first dozen or so bases of a sequence are
                                  vector, and should be excluded from
                                  consideration. The value for this parameter
                                  has the form
                                  (start),(end)
                                  where (start) is the index of the first base
                                  to consider, and (end) is the last in the
                                  primer-picking region.
   -target             range      If one or more Targets is specified then a
                                  legal primer pair must flank at least one of
                                  them. A Target might be a simple sequence
                                  repeat site (for example a CA repeat) or a
                                  single-base-pair polymorphism. The value
                                  should be a space-separated list of
                                  (start),(end)
                                  pairs where (start) is the index of the
                                  first base of a Target, and (end) is the
                                  last
                                  E.g. 50,51 requires primers to surround the
                                  2 bases at positions 50 and 51.
   -excludedregion     range      Primer oligos may not overlap any region
                                  specified in 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 the excluded region, and and
                                  (end) is the last. This tag is useful for
                                  tasks such as excluding regions of low
                                  sequence quality or for excluding regions
                                  containing repetitive elements such as ALUs
                                  or LINEs.
                                  E.g. 401,407 68,70 forbids selection of
                                  primers in the 7 bases starting at 401 and
                                  the 3 bases at 68.
   -forwardinput       string     The sequence of a forward primer to check
                                  and around which to design reverse primers
                                  and optional internal oligos. Must be a
                                  substring of SEQUENCE.
   -reverseinput       string     The sequence of a reverse primer to check
                                  and around which to design forward primers
                                  and optional internal oligos. Must be a
                                  substring of the reverse strand of SEQUENCE.
*  -gcclamp            integer    Require the specified number of consecutive
                                  Gs and Cs at the 3' end of both the forward
                                  and reverse primer. (This parameter has no
                                  effect on the internal oligo if one is
                                  requested.)
*  -osize              integer    Optimum length (in bases) of a primer oligo.
                                  Primer3 will attempt to pick primers close
                                  to this length.
*  -minsize            integer    Minimum acceptable length of a primer. Must
                                  be greater than 0 and less than or equal to
                                  MAX-SIZE.
*  -maxsize            integer    Maximum acceptable length (in bases) of a
                                  primer. Currently this parameter cannot be
                                  larger than 35. This limit is governed by
                                  the maximum oligo size for which Primer3's
                                  melting-temperature is valid.
*  -otm                float      Optimum melting temperature(Celsius) for a
                                  primer oligo. Primer3 will try to pick
                                  primers with melting temperatures 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.
*  -mintm              float      Minimum acceptable melting
                                  temperature(Celsius) for a primer oligo.
*  -maxtm              float      Maximum acceptable melting
                                  temperature(Celsius) for a primer oligo.
*  -maxdifftm          float      Maximum acceptable (unsigned) difference
                                  between the melting temperatures of the
                                  forward and reverse primers.
*  -ogcpercent         float      Primer optimum GC percent.
*  -mingc              float      Minimum allowable percentage of Gs and Cs in
                                  any primer.
*  -maxgc              float      Maximum allowable percentage of Gs and Cs in
                                  any primer generated by Primer.
*  -saltconc           float      The millimolar concentration of salt
                                  (usually KCl) in the PCR. Primer3 uses this
                                  argument to calculate oligo melting
                                  temperatures.
*  -dnaconc            float      The nanomolar concentration of annealing
                                  oligos in the PCR. Primer3 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.
*  -maxpolyx           integer    The maximum allowable length of a
                                  mononucleotide repeat in a primer, for
                                  example AAAAAA.
*  -productosize       integer    The optimum size for the PCR product. 0
                                  indicates that there is no optimum product
                                  size.
*  -productsizerange   range      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.
                                  Primer3 favors ranges to the left side of
                                  the parameter string. Primer3 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 Primer3 return primers in a
                                  subsequent range.
*  -productotm         float      The optimum melting temperature for the PCR
                                  product. 0 indicates that there is no
                                  optimum temperature.
*  -productmintm       float      The minimum allowed melting temperature of