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引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS)  

2011-11-25 09:00:21|  分类: 生物信息分析 |  标签: |举报 |字号 订阅

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BACKGROUND INFORMATION: For sites describing PCR theory, as well as companies marketing PCR products you might want to begin by visiting Horizon Press. For PCR techniques see PCRlink.com.  

There are several excellent sites for designing PCR primers:

red_bullet.gif (914 bytes) Primer3: WWW primer tool (University of Massachusetts Medical School, U.S.A.) This site has a very powerful PCR primer design program permitting one considerable control over the nature of the primers, including size of product desired, primer size and Tm range, and presence/absence of a 3’-GC clamp.
red_bullet.gif (914 bytes) GeneFisher - Interactive PCR Primer Design (Universitat Bielefeld, Germany) - a very good site allowing great control over primer design.
red_bullet.gif (914 bytes)PCR Now (Computational Biology Group, PathoGene, Southwestern Medical Center, U.S.A.) - created to design Real-Time Polymerase Chain Reaction (RT-PCR) primers for any number of user-defined coding sequences. Great control over primer properties. If you are interested in designing primers specific to published organismal or viral genes see the related site PathoGene.
red_bullet.gif (914 bytes) Primer3Plus - a new improved web interface to the popular Primer3 primer design program (Reference: A. Untergasser et al. 2007. Nucl. Acids Res. 35(Web Server issue):W71-W74)
red_bullet.gif (914 bytes) OligoCalc: an online oligonucleotide properties calculator - (Reference: W.A. Kibbe. 2007. Nucl. Acids Res. 35(Web Server issue):W43-W46)
red_bullet.gif (914 bytes) Primer-BLAST was developed at NCBI to help users make primers that are specific  to the input PCR template.  It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database.  The blast results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the input template.

red_bullet.gif (914 bytes) JOG 1.01 Javascript Oligonucleotide Generator (R.D. Mosteller) - will generate Fixed length and composition or Random length and composition oligonucleotides.

red_bullet.gif (914 bytes) RAPD-primer generator (J.W?stemeyer, Institute of General Microbiology and Microbial Genetics, Germany)

PCR primers based upon protein sequence:

red_bullet.gif (914 bytes) If you has the protein  sequence and  want the DNA sequence the best sites are Reverse Translate a Protein (Colorado State, U.S.A.) or the Java tool Protein backtranslation (Entelechon, Germany).  This site provides one with a wealth of options include organism-specific codon usage.  If you are interested in changing a specific amino acid into another you should consult Reverse Translator (EMBL).  One other site is CODEHOP (Fred Hutchinson Cancer Research Center, Washington, U.S.A.). The acronym is from COnsensus-DEgenerate Hybrid Oligonucleotide Primers, and is used to design primers based upon multiple sequence alignments.

PCR primers based upon multialignments:

red_bullet.gif (914 bytes) Primaclade (Molecular Systematics Laboratory at the University of Missouri - St. Louis, U.S.A.) - this application accepts a multiple species nucleotide alignment file as input and identifies a set of PCR primers that will bind across the alignment.
red_bullet.gif (914 bytes) PriFi - upload a file containing Fasta-formatted DNA sequences or alternatively a *.aln file, select the control one  wants over the primer design from an extensive list and press "Find primers in alignment." (Reference: J. Fredslund et al. 2005. Nuc. Acids Res. 33: W516-W520).

Genomic scale primers: (N.B. also see the JAVA page for additional downloadable programs)

red_bullet.gif (914 bytes) The PCR Suite (Klinische Genetica, Erasmus MC Rotterdam, Netherlands) - this is a suite of four programs based upon Primer3 for genomic primer design.  All offer considerable control on primer properties:

引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS) - 喜欢吃桃子 - wangyufeng的博客 Overlapping_Primers - creates multiple overlapping PCR products in one sequence.
引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS) - 喜欢吃桃子 - wangyufeng的博客
Genomic_Primers - designs primers around exons in genomic sequence. All you need is a GenBank file containing your  gene.
引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS) - 喜欢吃桃子 - wangyufeng的博客
SNP_Primers - designs primers around every SNP in a GenBank file.
引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS) - 喜欢吃桃子 - wangyufeng的博客
cDNA_Primers - designs primers around open reading frames. Simply upload a GenBank file containing your genes.

red_bullet.gif (914 bytes) MuPlex: multi-objective multiplex PCR assay design - designed for large-scale multiplex PCR assay design in an automated high-throughput environment, where high coverage is required. (Reference: J. Rachlin et al. 2005. Nucl. Acids Res. 33: W544-W547).

Overlapping primer sets:

red_bullet.gif (914 bytes) Overlapping Primersets - This software is based on the Primer3 program developed by the Whitehead Institute (see above). A closely related site is Multiple Primer Design with Primer 3

red_bullet.gif              (914 bytes)GenoFragis a software package to design primers optimized for whole genome scanning by long-range PCR. It was developed for the analysis of Staphylococcus aureus genome plasticity by whole genome amplification in ~10 kb-long fragments. Site is in French. (Reference: N. Ben Zakour et al. 2004. Nucl. Acids Res. 32: 17-24)

Short interfering RNA (siRNA) design:

red_bullet.gif (914 bytes) SiRNA Selector - Small interfering RNA (siRNA) guides sequence-specific degradation of the homologous mRNA, thus producing "knock-down" cells. siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. The program evaluates siRNA functionality and specificity. (Reference: N. Levenkova et al. (2003) Bioinformatics 2004 20: 430-432). Other similar programs are  siRNA Target Designer  (Promega, U.S.A.); the similarly named  siRNA Target Finder(Ambion, U.S.A.) and siRNA Target Finder (GenScript USA Inc.).

引物设计不求人:常用引物设计工具大全(DESIGN PCR PRIMERS) - 喜欢吃桃子 - wangyufeng的博客 siRNA Design Software - compares existing design tools, including those listed above. They also attempt to improve the MPI principles and existing tools by an algorithm that can filter ineffective siRNAs. The algorithm is based on some new observations on the secondary structure. (Reference: S. M. Yiu et al. (2004) Bioinformatics 21: 144-151).

Realtime PCR primer design:

red_bullet.gif (914 bytes) RealTimeDesign (Biosearch Technoloogies) - free but requires registration.

red_bullet.gif (914 bytes) QuantPrimeis a flexible program for reliable primer design for use in larger qPCR experiments. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. (Reference: S. Arvidsson et al. 2008. BMC Bioinformatics  9:465)

red_bullet.gif (914 bytes)  Real-time PCR (TaqMan) Primer Design (GenScript USA Inc.)

For additional physicochemical data on the primers the following six sites are useful:

red_bullet.gif (914 bytes) NetPrimer (Premier Biosoft International, U.S.A.) - In my opinion the best site since it provides one with Tm, thermodynamic properties and most stable hairpin & dimers.BUT it takes a while for the program to load.  

red_bullet.gif (914 bytes) dnaMATE - calculates a consensus Tm) for short DNA sequence (16-30 nts) using a merged method that is based on three different thermodynamic tables. The consensus Tm value is a robust and accurate estimation of melting temperature for short DNA sequences of practical application in molecular biology. Accuracy benchmarks using all experimental data available indicate that the consensus Tm prediction errors will be within 5 ?C from the experimental value in 89% of the cases. (Reference: A. Panjkovich et al. 2005. Nucl. Acids Res. 33:  W570-W572.).

red_bullet.gif (914 bytes) OligoAnalyzer (Integrated DNA Technologies, Inc., U.S.A.) - in addition to hairpin and self-dimer analysis of existing primers this site provides one with the opportunity to BLAST the sequence against NCBI's database and measure the impact of incorporating 5'-modifications into the sequence. The oligos can then be ordered directly.
red_bullet.gif (914 bytes) Another excellent site is Oligonucleotide Properties Calculator (Northwestern University Medical School, Chicago, U.S.A.) which provides one with detailed information on the calculations.  Also permits analysis of  6-FAM, HEX, or TAMRA-labelled oligos. 
red_bullet.gif (914 bytes) Biopolymer Calculator (Yale University, U.S.A.) - Not yet functional.
red_bullet.gif (914 bytes) Melting: enthalopy, entropy and melting temperature (N. Le Novere, Pasteur Institute, Paris, France).

Introduction of silent mutations:

red_bullet.gif (914 bytes) WatCut (Michael Palmer, University of Waterloo, Canada) - takes an oligonucleotide and introduces silent mutations in potential restriction sites such that the amino acid sequence of the protein is unaltered. 

When you are ready to set-up your PCR reaction see:

red_bullet.gif (914 bytes) PCR Box Titration Calculator (Allotron Biosensor Corporation) - for figuring out the amounts of each reagent to use in a two-dimensional box titration for PCR.  For standard PCR reactions adjust volume, and change "row" and "column" number to "1", click on all the "top" or "bottom" and "done".

red_bullet.gif (914 bytes) PCR Reaction Mixture Setup (R. Kalendar, University of Helsinki, Finland) - very nice site.

Primer presentation on the DNA sequence:

red_bullet.gif (914 bytes) Sequence Extractor (Paul Stothard) - generates a clickable restriction map and PCR primer map of a DNA sequence (Accepted formats are: raw, GenBank, EMBL, and FASTA) offering a great deal of control on output. Protein translations and intron/exon boundaries are also shown. Use Sequence Extractor to build DNA constructs in silico.

via:http://molbiol-tools.ca/PCR.htm

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