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below (5'-&3', string of ACGTNacgtn -- other letters treated as N -- numbers and blanks ignored). FASTA format ok. Please N-out undesirable sequence (vector, ALUs, LINEs, etc.) or use a
NONEHUMANRODENT_AND_SIMPLERODENTDROSOPHILA
Pick left primer or use left primer below.
Pick hybridization probe (internal oligo) or use oligo below.
Pick right primer or use right primer below (5'-&3' on opposite strand).
A string to identify your output.
E.g. 50,2 requires primers to surround the 2 bases at positions 50 and 51. Or mark the
with [ and ]: e.g. ...ATCT[CCCC]TCAT.. means that primers must flank the central CCCC.
E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the
with & and &: e.g. ...ATCT&CCCC&TCAT.. forbids primers in the central CCCC.
General Primer Picking Conditions
Do not treat ambiguity codes in libraries as consensus
Other Per-Sequence Inputs
E.g. 20,400: only pick primers in the 400 base region starting at position 20. Or use { and } in the
to mark the beginning and end of the included region: e.g. in ATC{TTC...TCT}AT the included region is TTC...TCT.
Objective Function Penalty Weights for Primers
Objective Function Penalty Weights for Primer Pairs
NONEHUMANRODENT_AND_SIMPLERODENTDROSOPHILA
Objective Function Penalty Weights for Hyb Oligos (Internal Oligos)
Copyright (c) 98,01,2004 Whitehead Institute for Biomedical Research. All rights reserved.
Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:
Redistributions must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution. Redistributions of source code must also reproduce this information in the source code itself.
If the program is modified, redistributions must include a notice (in the same places as above) indicating that the redistributed program is not identical to the version distributed by Whitehead Institute.
All advertising materials mentioning features or use of this software must display the following acknowledgment:
This product includes software developed by the Whitehead Institute for Biomedical Research.
The name of the Whitehead Institute may not be used to endorse or promote products derived from this software without specific prior written permission. We also request that use of this software be cited in publications as
and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386THIS SOFTWARE IS PROVIDED BY THE WHITEHEAD INSTITUTE ``AS IS'' AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE WHITEHEAD INSTITUTE BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
AcknowledgmentsThe development of Primer3 and the Primer3 web site was funded by
and by the
under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander).
We gratefully acknowledge the support of Digital Equipment Corporation, which provided the Alphas which were used for much of the development of Primer3, and of Centerline Software, Inc., whose TestCenter memory-error, -leak, and test-coverage checker we use regularly to discover and correct otherwise latent errors in Primer3.
Primer3's design is heavily based on an earlier implementation of a similar program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). Lincoln Stein championed the idea of making the Primer3 engine a software component.
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Primer3在线引物设计工具使用方法
Primer3在线引物设计工具使用方法
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引物设计 开始之前：其实非常简单，不需要你下载任何软件
，但是你得有一台电脑能上网。当然，最重要的是，你要很清楚用于做引物的模板序列，至于怎么找模板序列，不再本次讨论范围。另外，要先对PCR
目的序列的长度有个大致估计。
第一步：找到Primer 3的站点。你不用记住这个站点，但是要记住“Primer3”这个词，然后打开GOOGLE首页，输入Primer3，跳出来的第一个项目就是了“Primer3 Input 0.4.0 ”，网址是http://frodo.wi.mit.edu/。
第二步：贴上模板序列
。进入Primer3站点，可以看到一个引物设计
的界面。(如下图)在“Paste source sequence below (5'-&3'…”下面的大空框里面把你的模板序列粘帖进去。注意是5'-&3'方向的，数字或者空格都没关系，软件会自动过滤的。
第三步：重要参数设定。首先是“Product Size Ranges ”，如果你不希望软件给你随便做的话，首先要调整的就是这个参数。默认的参数实际上是从100到1000，这个你得自己改，如果你希望产物的大小符合你的预期，尽可能把范围改小，比如480-500，具体看情况调整。第二个参数是“Primer Size”，默认值一般可以用，但是，当你用熟了这个软件，你自己就知道该怎么改了。第三个参数是“Primer Tm ”这个和Primer Size差不多。
第四步：Pick primers： 点一下这个按钮，符合你大小预期的primer就出来了，看看Primer3 Output的界面，多么漂亮!你要的primer出来了，而且有primer在序列上的位置比对图，还要primer本身的信息，包括位置，长度，Tm，GC含量，任何位置互补碱基数，3'端互补碱基数，以及引物序列，(注意，下游引物是5'-&3')，还要产物大小，两引物间任意互补碱基数，两引物间3'端互补碱基数等。如果引物尚在参数设定的范围内，但还不是最佳，将会给出警告。
比如(随便举例，本人在做一个超长引物)：
WARNING: Left primer is unacceptable: High 3' stability
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 1 33 69.02 45.45 6.00 1.00 TAATACGACTCACTATAGGGGTGAAAGACTGCC
RIGHT PRIMER .86 29.41 6.00 2.00 AAAGGGTTAATTTGCATGCTTTATTTACACACAT
SEQUENCE SIZE: 1388
INCLUDED REGION SIZE: 1388
PRODUCT SIZE: 1388, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
第五步：引物设计检验：可以仅仅设计一对引物，只要在Pick left primer或者Pick right primer前面的勾勾掉一个就可以。也可以自己定义引物，放在Primer框里，(注意，下游引物的书写反向仍然是5'-&3')如果符合设定的条件，软件将对给出引物评分，同时给出警告信息，根据警告信息可以适当对自定义引物做些调整即可，警告信息也让你做实验的时候心中有数。对于文献发表的引物，最好都要检查一下，这样就可以避免被别人有意无意误导。至于RT-PCR
所用的引物，最好是使得产物跨过内含子，这样避免潜在DNA对RT-PCR
的干扰。实际做引物的时候，要把内含子都去掉，仅仅把外显子序列放入源序列框中，并且通过自定义引物设计的方法，使上下游引物分别全部或者部分落在不同外显子上。至于如何快速识别内含子和外显子以及电子PCR，将另做说明。
至于设计出来的引物的实际效果，根据经验，一般情况下都能做到PCR一次成功，也许随便那一段序列做引物也能达到很好的效果，但是不管怎么说，软件做引物可以让自己心中有数。
相关实验方法
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你可能喜欢The information below is meant to provide an "easy" means of locating and downloading the code/binaries you need to run Primer3.
As such, we have provided links to help you reach the most recent versions of the software.
ALL downloads including older versions of the software are available from the .
Primer3 (the C code)
Release 2.3.7 (February 18, 2016)
Web Interfaces
Primer3Web
Release 4.0.0 (Jan 24, 2013)
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