primer3.itable

emboss的linux版本的源代码

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<th align="left" colspan=2>Mandatory qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
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<td>[-sequence]<br>(Parameter 1)</td>
<td>The sequence from which to choose primers. The sequence must be presented 5' to 3'</td>
<td>Readable sequence(s)</td>
<td><b>Required</b></td>
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<td>[-outfile]<br>(Parameter 2)</td>
<td>Output file name</td>
<td>Output file</td>
<td><i>&lt;sequence&gt;</i>.primer3</td>
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<th align="left" colspan=2>Optional qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
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<td>-task</td>
<td>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.</td>
<td><table><tr><td>0</td> <td><i>(Pick PCR primers)</i></td></tr><tr><td>1</td> <td><i>(Pick PCR primers and hybridization probe)</i></td></tr><tr><td>2</td> <td><i>(Pick forward primer only)</i></td></tr><tr><td>3</td> <td><i>(Pick reverse primer only)</i></td></tr><tr><td>4</td> <td><i>(Pick hybridization probe only)</i></td></tr></table></td>
<td>0</td>
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<td>-numreturn</td>
<td>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.</td>
<td>Integer 0 or more</td>
<td>5</td>
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<td>-includedregion</td>
<td>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.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
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<td>-target</td>
<td>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.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
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<td>-excludedregion</td>
<td>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.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
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<td>-forwardinput</td>
<td>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.</td>
<td>Any string is accepted</td>
<td><i>An empty string is accepted</i></td>
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<td>-reverseinput</td>
<td>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.</td>
<td>Any string is accepted</td>
<td><i>An empty string is accepted</i></td>
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<td>-gcclamp</td>
<td>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.)</td>
<td>Integer 0 or more</td>
<td>0</td>
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<td>-osize</td>
<td>Optimum length (in bases) of a primer oligo. Primer3 will attempt to pick primers close to this length.</td>
<td>Integer 1 or more</td>
<td>20</td>
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<td>-minsize</td>
<td>Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to MAX-SIZE.</td>
<td>Integer 1 or more</td>
<td>18</td>
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<td>-maxsize</td>
<td>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.</td>
<td>Integer up to 35</td>
<td>27</td>
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<td>-otm</td>
<td>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.</td>
<td>Any integer value</td>
<td>60.0</td>
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<td>-mintm</td>
<td>Minimum acceptable melting temperature(Celsius) for a primer oligo.</td>
<td>Any integer value</td>
<td>57.0</td>
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<td>-maxtm</td>
<td>Maximum acceptable melting temperature(Celsius) for a primer oligo.</td>
<td>Any integer value</td>
<td>63.0</td>
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<td>-maxdifftm</td>
<td>Maximum acceptable (unsigned) difference between the melting temperatures of the forward and reverse primers.</td>
<td>Any integer value</td>
<td>100.0</td>
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<td>-ogcpercent</td>
<td>Primer optimum GC percent.</td>
<td>Any integer value</td>
<td>50.0</td>
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<td>-mingc</td>
<td>Minimum allowable percentage of Gs and Cs in any primer.</td>
<td>Any integer value</td>
<td>20.0</td>
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<td>-maxgc</td>
<td>Maximum allowable percentage of Gs and Cs in any primer generated by Primer.</td>
<td>Any integer value</td>
<td>80.0</td>
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<td>-saltconc</td>
<td>The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures.</td>
<td>Any integer value</td>
<td>50.0</td>
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<td>-dnaconc</td>
<td>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.</td>
<td>Any integer value</td>
<td>50.0</td>
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<td>-maxpolyx</td>
<td>The maximum allowable length of a mononucleotide repeat in a primer, for example AAAAAA.</td>
<td>Integer 0 or more</td>
<td>5</td>
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<td>-productosize</td>
<td>The optimum size for the PCR product. 0 indicates that there is no optimum product size.</td>
<td>Integer 0 or more</td>
<td>200</td>
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<td>-productsizerange</td>
<td>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.</td>
<td>Sequence range</td>
<td>100-300</td>
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<td>-productotm</td>
<td>The optimum melting temperature for the PCR product. 0 indicates that there is no optimum temperature.</td>
<td>Any integer value</td>
<td>0.0</td>
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<td>-productmintm</td>
<td>The minimum allowed melting temperature of the amplicon. Please see the documentation on the maximum melting temperature of the product for details.</td>
<td>Any integer value</td>
<td>-1000000.0</td>
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<td>-productmaxtm</td>
<td>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.</td>
<td>Any integer value</td>
<td>1000000.0</td>
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<td>-oligoexcludedregion</td>
<td>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.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
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