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