Primer
Design Algorithms, Databases &
Resources
General Priming
Strategies
Real-time PCR primer and probe
databases:
- RTPrimerDB:
the Real-Time PCR primer and probe database
PATTYN, F.,
SPELEMAN, F., DE PAEPE A. & VANDESOMPELE, J.
Nucleic Acids Research, 31(1): 122-123
The real-time
polymerase chain
reaction (PCR) methodology has become increasingly popular for nucleic
acids detection and/or quantification. As primer/probe design and
experimental evaluation is time-consuming, we developed a public
database application for the storage and retrieval of validated
real-time PCR primer and probe sequence records. The integrity and
accuracy of the data are maintained by linking to and querying other
reference databases. RTPrimerDB provides free public access through the
Web to perform queries and submit user based information. Primer/probe
records can be searched for by official gene symbol, nucleotide
sequence, type of application, detection chemistry, LocusLink or Single
Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each
record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve
additional information on the gene/SNP for which the primers/probes are
designed. Currently, the database contains primer/probe records for
human, mouse, rat, fruit fly and zebrafish, and all current detection
chemistries such as intercalating dyes (SYBR Green I), hydrolysis
probes (Taqman), adjacent hybridizations probes and molecular beacons.
Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st
- RTPrimerDB: the real-time PCR primer and
probe database, major update 2006
Pattyn F, Robbrecht P, De Paepe A, Speleman F, Vandesompele J.
Center for Medical Genetics Ghent (CMGG), Ghent University Hospital, De
Pintelaan 185, 9000 Ghent, Belgium.
Nucleic Acids Res. 2006 Jan 1;34(Database issue): D684-688
The RTPrimerDB
(http://medgen.ugent.be/rtprimerdb) project provides a freely
accessible data retrieval system and an in silico assay evaluation
pipeline for real-time quantitative PCR assays. Over the last year the
number of user submitted assays has grown to 3500. Data conveyance from
Entrez Gene by establishing an assay-to-gene relationship enables the
addition of new primer assays for one of the 1.5 million different
genes from 2300 species stored in the system. Easy access to the primer
and probe data is possible by using multiple search criteria. Assay
reports contain gene information, assay details (such as
oligonucleotide sequences, detection chemistry and reaction
conditions), publication information, users' experimental evaluation
feedback and submitter's contact details. Gene expression assays are
extended with a scalable assay viewer that provides detailed
information on the alignment of primer and probe sequences on the known
transcript variants of a gene, along with Single Nucleotide
Polymorphisms (SNP) positions and peptide domain information.
Furthermore, an mfold module is implemented to predict the secondary
structure of the amplicon sequence, as this has been reported to impact
the efficiency of the PCR. RTPrimerDB is also extended with an in
silico analysis pipeline to streamline the evaluation of custom
designed primer and probe sequences prior to ordering and experimental
evaluation. In a secured environment, the pipeline performs automated
BLAST specificity searches, mfold secondary structure prediction, SNP
or plain sequence error identification, and graphical visualization of
the aligned primer and probe sequences on the target gene.
- RTPrimerDB: the portal for real-time PCR
primers and probes
Lefever S, Vandesompele J, Speleman F, Pattyn F.
Center for Medical Genetics, Ghent University Hospital, De Pintelaan
185, 9000 Gent, Belgium.
Nucleic Acids Res. 2009 Jan;37(Database issue): D942-945
RTPrimerDB http://www.rtprimerdb.org
is a freely accessible database and analysis tool for
real-time quantitative PCR assays. RTPrimerDB includes records with
user submitted assays that are linked to genome information from
reference databases and quality controlled using an in silico assay
evaluation system. The primer evaluation tools intended to assess the
specificity and to detect features that could negatively affect the
amplification efficiency are combined into a pipeline to test
custom-designed primer and probe sequences. An improved user feedback
system guides users and submitters to enter practical remarks and
details about experimental evaluation analyses. The database is linked
with reference databases to allow the submission of assays for all
genes and organisms officially registered in Entrez Gene and RefSeq.
Records in RTPrimerDB are assigned unique and stable identifiers. The
content is provided via an interactive web-based search system and is
available for download in the recently developed RDML format and as
bulk export file. RTPrimerDB is a one-stop portal for high-quality and
highly annotated real-time PCR assays.
- The
BiSearch web server
Aranyi
T, Varadi A, Simon I, Tusnady GE.
BMC Bioinformatics. 2006 ;7: 431.
lnstitute
of Enzymology, BRC, HAS, H-1113 Karolina ut 29, Budapest, Hungary.
BACKGROUND:
A large number of PCR primer-design softwares are available online.
However, only very
few of them can be used for the design of primers to amplify
bisulfite-treated DNA
templates, necessary to determine genomic DNA methylation profiles.
Indeed, the
number of studies on bisulfite-treated templates exponentially
increases as determining DNA methylation becomes more important in the
diagnosis of
cancers. Bisulfite-treated DNA is difficult to amplify since undesired
PCR
products are often amplified due to the increased sequence redundancy
after the
chemical conversion. In order to increase the efficiency of PCR
primer-design, we
have developed BiSearch web server, an online primer-design tool
for both bisulfite-treated and native DNA templates. RESULTS: The
web tool is
composed of a primer-design and an electronic PCR (ePCR) algorithm. The
completely reformulated ePCR module detects potential mispriming sites
as well as
undesired PCR products on both cDNA and native or bisulfite-treated
genomic DNA libraries. Due to the new algorithm of the current version,
the ePCR
module became approximately hundred times faster than the previous one
and gave
the best performance when compared to other web based tools. This
high-speed ePCR analysis made possible the development of the new
option of
high-throughput primer screening. BiSearch web server can be used for
academic researchers
at the http://bisearch.enzim.hu
site. CONCLUSION: BiSearch web server is a
useful tool for primer-design for any DNA template and especially for
bisulfite-treated genomes. The ePCR tool for fast detection of
mispriming sites and
alternative PCR products in cDNA libraries and native or
bisulfite-treated
genomes are the unique features of the new version of BiSearch software.
-
BiSearch: primer-design and search tool for
PCR on bisulfite-treated genomes
Tusnady GE, Simon I, Varadi A, Aranyi T.
Nucleic Acids Res. 2005 Jan 13;33(1):e9.
Institute of Enzymology, BRC, Hungarian Academy of
Sciences H-1113 Budapest, Karolina ut 29, Hungary.
Bisulfite genomic sequencing is the most widely used
technique to analyze the 5-methylation of cytosines,
the prevalent covalent DNA modification in mammals. The
process is based on the selective transformation of unmethylated
cytosines to uridines. Then, the investigated genomic
regions are PCR amplified, subcloned and sequenced.
During sequencing, the initially unmethylated cytosines are detected
as thymines. The efficacy of bisulfite PCR is generally low; mispriming
and non-specific amplification often occurs
due to the T richness of the target sequences. In order
to ameliorate the efficiency of PCR, we developed a new primer-design
software called BiSearch, available on the World Wide Web. It has the
unique property of analyzing the primer pairs for mispriming sites on
the bisulfite-treated genome and determines potential
non-specific amplification products with a new search
algorithm. The options of primer-design and analysis for
mispriming sites can be used sequentially or separately, both on bisulfite-treated
and untreated sequences. In silico and in vitro tests of the software
suggest that new PCR strategies may increase the efficiency of the amplification.
- PRaTo: A web-tool to select optimal primer
pairs for qPCR
Alberto Nonis, Marco Scortegagna, Alessandro Nonis, Benedetto Ruperti
Biochemical and Biophysical Research Communications 415 (2011) 707–708
An essential
pre-requisite to perform sound quantitative real-time
polymerase chain reaction (qPCR) assays is to design outstanding primer
pairs. This means they must have a good efficiency and be not prone to
produce multiple amplicons or primer dimer products. To circumvent
these issues, several softwares are available to help primer design.
Although satisfactory computer-aided primer design tools are available
for standard PCR, less efforts were done to provide specific methods
for selection of optimal primer pairs for qPCR. We have developed PRaTo
a web-based tool that enables checking and ranking of primers pairs for
their attitude to perform optimally and reliably when used in qPCR
experiments. PRaTo is available at http://prato.daapv.unipd.it
-
The
ability to select short DNA oligonucleotide sequences capable of
binding solely to their intended target is of great importance in
developing nucleic acid based detection
technologies. Applications such as multiplex PCR rely on primers
binding to unique regions in a genome. Competing side reactions with
other primer pairs or template DNA decrease PCR efficiency: Freely
available primer design software such as Primer3 screens for potential
hairpin and primer-dimer interactions while selecting a single primer
pair. The development of multiplex PCR assays (in the range of 5 to 20
loci) requires the screening of all primer pairs for potential
cross-reactivity. However, a logistical problem results due to the
number of total number of comparisons required. Comparing the
candidate oligomers rapidly for potential cross-reactivity reduces
overall assay devlelopment time. Here we report the application of a
familiar sliding algorithm for comparing two strands of DNA in an
overlapping fashion. The algorithm has been employed in a software
package wherein the user can compare user-defined threshold. Additional
criteria of predicted melting temperature (Tm) and free energy of
melting (deltaG) are included for further ranking. Sodium counterion
and total stand concentrations can be adjusted for the Tm
and deltaG calculations. primer set for a 10-plex assay (20
total
primer sequences) results in 210 primer-primer combinations that must
be
screened. The ability to screen sets of multiple sequences in a single
computational
run. After the screening is completed, a score is assigned to potential
duplex
interactions exceeding a The predicted
interactions are saved in a text file for further evaluation.
- A PCR primer bank for quantitative gene
expression analysis
Wang X, Seed B.
Department of Molecular Biology, Massachusetts General Hospital, 50
Blossom Street, Boston, MA 02114, USA.
Nucleic Acids Res. 2003 Dec 15;31(24):e154
Although gene
expression profiling by microarray analysis is a useful tool for
assessing global levels of transcriptional activity, variability
associated with the data sets usually requires that observed
differences be validated by some other method, such as real-time
quantitative polymerase chain reaction (real-time PCR). However,
non-specific amplification of non-target genes is frequently observed
in the latter, confounding the analysis in approximately 40% of
real-time PCR attempts when primer-specific labels are not used. Here
we present an experimentally validated algorithm for the identification
of transcript-specific PCR primers on a genomic scale that can be
applied to real-time PCR with sequence-independent detection methods.
An online database, PrimerBank, has been created for researchers to
retrieve primer information for their genes of interest. PrimerBank
currently contains 147 404 primers encompassing most known human and
mouse genes. The primer design algorithm has been tested by
conventional and real-time PCR for a subset of 112 primer pairs with a
success rate of 98.2%.
- A comprehensive collection of
experimentally validated primers for Polymerase Chain Reaction
quantitation of murine transcript abundance
Spandidos A, Wang X, Wang H, Dragnev S, Thurber T, Seed B.
Center for Computational and Integrative Biology, Massachusetts General
Hospital, MA, USA
BMC Genomics. 2008 Dec 24;9:633.
BACKGROUND: Quantitative polymerase chain reaction (QPCR) is a widely
applied analytical method for the accurate determination of transcript
abundance. Primers for QPCR have been designed on a genomic scale but
non-specific amplification of non-target genes has frequently been a
problem. Although several online databases have been created for the
storage and retrieval of experimentally validated primers, only a few
thousand primer pairs are currently present in existing databases and
the primers are not designed for use under a common PCR thermal profile.
RESULTS: We previously reported the implementation of an algorithm to
predict PCR primers for most known human and mouse genes. We now report
the use of that resource to identify 17483 pairs of primers that have
been experimentally verified to amplify unique sequences corresponding
to distinct murine transcripts. The primer pairs have been validated by
gel electrophoresis, DNA sequence analysis and thermal denaturation
profile. In addition to the validation studies, we have determined the
uniformity of amplification using the primers and the technical
reproducibility of the QPCR reaction using the popular and inexpensive
SYBR Green I detection method.
CONCLUSION: We have identified an experimentally validated collection
of murine primer pairs for PCR and QPCR which can be used under a
common PCR thermal profile, allowing the evaluation of transcript
abundance of a large number of genes in parallel. This feature is
increasingly attractive for confirming and/or making more precise data
trends observed from experiments performed with DNA microarrays.
- PrimerBank: a resource of human and mouse
PCR primer pairs for gene expression detection and quantification
Spandidos A, Wang X, Wang H, Seed B.
Department of Genetics, Harvard Medical School, Center for
Computational and Integrative Biology, Massachusetts General Hospital,
Boston, MA 02114-2790, USA.
Nucleic Acids Res. 2010 Jan;38(Database issue): D792-799
PrimerBank http://pga.mgh.harvard.edu/primerbank/
is a public resource for the retrieval of human and mouse
primer pairs for gene expression analysis by PCR and Quantitative PCR
(QPCR). A total of 306,800 primers covering most known human and mouse
genes can be accessed from the PrimerBank database, together with
information on these primers such as T(m), location on the transcript
and amplicon size. For each gene, at least one primer pair has been
designed and in many cases alternative primer pairs exist. Primers have
been designed to work under the same PCR conditions, thus facilitating
high-throughput QPCR. There are several ways to search for primers for
the gene(s) of interest, such as by: GenBank accession number, NCBI
protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol
or gene description (keyword). In all, 26,855 primer pairs covering
most known mouse genes have been experimentally validated by QPCR,
agarose gel analysis, sequencing and BLAST, and all validation data can
be freely accessed from the PrimerBank web site.
- PrimerBank: a PCR primer database for
quantitative gene expression analysis, 2012 update
Wang X, Spandidos A, Wang H, Seed B.
Department of Radiation Oncology, Washington University School of
Medicine, 4511 Forest Park Ave, Saint Louis, MO 63108, USA.
Nucleic Acids Res. 2012 Jan;40(Database
issue):D1144-9
Optimization of
primer sequences for polymerase chain reaction (PCR) and quantitative
PCR (qPCR) and reaction conditions remains an experimental challenge.
We have developed a resource, PrimerBank, which contains primers that
can be used for PCR and qPCR under stringent and allele-invariant
amplification conditions. A distinguishing feature of PrimerBank is the
experimental validation of primer pairs covering most known mouse
genes. Here, we describe a major update of PrimerBank that includes the
design of new primers covering 17,076 and 18,086 genes for the human
and mouse species, respectively. As a result of this update, PrimerBank
contains 497,156 primers (an increase of 62% from the previous version)
that cover 36,928 human and mouse genes, corresponding to around 94% of
all known protein-coding gene sequences. An updated algorithm based on
our previous approach was used to design new primers using current
genomic information available from the National Center for
Biotechnology Information (NCBI). PrimerBank primers work under uniform
PCR conditions, and can be used for high-throughput or genome-wide
qPCR. Because of their broader linear dynamic range and greater
sensitivity, qPCR approaches are used to reanalyze changes in
expression suggested by exploratory technologies such as microarrays
and RNA-Seq. The primers and all experimental validation data can be
freely accessed from the PrimerBank website, http://pga.mgh.harvard.edu/primerbank/
Distribution
of all rejected human primer candidates by various
bioinformatics screening filters. Combined together, 99% of all primer
candidates were rejected by these screening filters.
- GETPrime: a gene- or transcript-specific
primer database for quantitative real-time PCR
Gubelmann C, Gattiker A, Massouras A, Hens K, David F, Decouttere F,
Rougemont J, Deplancke B.
Institute of Bio-engineering, School of Life Sciences, Laboratory of
Systems Biology and Genetics, Lausanne, Switzerland.
Database (Oxford). 2011 Sep 14;2011:bar040. Print 2011.
Database URL: http://deplanckelab.epfl.ch
The vast majority of
genes in humans and other organisms undergo alternative splicing, yet
the biological function of splice variants is still very poorly
understood in large part because of the lack of simple tools that can
map the expression profiles and patterns of these variants with high
sensitivity. High-throughput quantitative real-time polymerase chain
reaction (qPCR) is an ideal technique to accurately quantify nucleic
acid sequences including splice variants. However, currently available
primer design programs do not distinguish between splice variants and
also differ substantially in overall quality, functionality or
throughput mode. Here, we present GETPrime, a primer database supported
by a novel platform that uniquely combines and automates several
features critical for optimal qPCR primer design. These include the
consideration of all gene splice variants to enable either
gene-specific (covering the majority of splice variants) or
transcript-specific (covering one splice variant) expression profiling,
primer specificity validation, automated best primer pair selection
according to strict criteria and graphical visualization of the latter
primer pairs within their genomic context. GETPrime primers have been
extensively validated experimentally, demonstrating high transcript
specificity in complex samples. Thus, the free-access, user-friendly
GETPrime database allows fast primer retrieval and visualization for
genes or groups of genes of most common model organisms, and is
available at http://updepla1srv1.epfl.ch/getprime/
- methBLAST and methPrimerDB: web-tools for
PCR based methylation analysis
Pattyn F, Hoebeeck J, Robbrecht P, Michels E, De Paepe A, Bottu G,
Coornaert D, Herzog R, Speleman F, Vandesompele J.
Center for Medical Genetics, Ghent University Hospital, De Pintelaan
185, 9000 Ghent, Belgium.
BMC Bioinformatics. 2006 Nov 9;7:496.
BACKGROUND: DNA
methylation plays an important role in development and tumorigenesis by
epigenetic modification and silencing of critical genes. The
development of PCR-based methylation assays on bisulphite modified DNA
heralded a breakthrough in speed and sensitivity for gene methylation
analysis. Despite this technological advancement, these approaches
require a cumbersome gene by gene primer design and experimental
validation. Bisulphite DNA modification results in sequence alterations
(all unmethylated cytosines are converted into uracils) and a general
sequence complexity reduction as cytosines become underrepresented.
Consequently, standard BLAST sequence homology searches cannot be
applied to search for specific methylation primers.
RESULTS: To address
this problem we developed methBLAST, a sequence similarity search
program, based on the original BLAST algorithm but querying in silico
bisulphite modified genome sequences to evaluate oligonucleotide
sequence similarities. Apart from the primer specificity analysis tool,
we have also developed a public database termed methPrimerDB for the
storage and retrieval of validated PCR based methylation assays. The
web interface allows free public access to perform methBLAST searches
or database queries and to submit user based information. Database
records can be searched by gene symbol, nucleotide sequence, analytical
method used, Entrez Gene or methPrimerDB identifier, and submitter's
name. Each record contains a link to Entrez Gene and PubMed to retrieve
additional information on the gene, its genomic context and the article
in which the methylation assay was described. To assure and maintain
data integrity and accuracy, the database is linked to other reference
databases. Currently, the database contains primer records for the most
popular PCR-based methylation analysis methods to study human, mouse
and rat epigenetic modifications. methPrimerDB and methBLAST are
available at http://medgen.ugent.be/methprimerdb
and http://medgen.ugent.be/methblast
CONCLUSION: We have
developed two integrated and freely available web-tools for PCR based
methylation analysis. methBLAST allows in silico assessment of primer
specificity in PCR based methylation assays that can be stored in the
methPrimerDB database, which provides a search portal for validated
methylation assays.
- AtRTPrimer: database for Arabidopsis
genome-wide homogeneous and specific RT-PCR primer-pairs
Han S, Kim D.
Department of BioSystems, Korea Advanced Institute of Science and
Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic
of Korea
BMC Bioinformatics. 2006 Mar 30;7:179.
BACKGROUND: Primer design is a critical step in all types of RT-PCR
methods to ensure specificity and efficiency of a target amplicon.
However, most traditional primer design programs suggest primers on a
single template of limited genetic complexity. To provide researchers
with a sufficient number of pre-designed specific RT-PCR primer pairs
for whole genes in Arabidopsis, we aimed to construct a genome-wide
primer-pair database.
DESCRIPTION: We considered the homogeneous physical and chemical
properties of each primer (homogeneity) of a gene, non-specific binding
against all other known genes (specificity), and other possible
amplicons from its corresponding genomic DNA or similar cDNAs
(additional information). Then, we evaluated the reliability of our
database with selected primer pairs from 15 genes using conventional
and real time RT-PCR.
CONCLUSION: Approximately 97% of 28,952 genes investigated were finally
registered in AtRTPrimer. Unlike other freely available primer
databases for Arabidopsis thaliana, AtRTPrimer provides a large number
of reliable primer pairs for each gene so that researchers can perform
various types of RT-PCR experiments for their specific needs.
Furthermore, by experimentally evaluating our database, we made sure
that our database provides good starting primer pairs for Arabidopsis
researchers to perform various types of RT-PCR experiments.
Real-time PCR primer
design tools:
- RExPrimer: an integrated primer designing
tool increases PCR effectiveness by avoiding 3' SNP-in-primer and
mis-priming from structural variation
Piriyapongsa J, Ngamphiw C, Assawamakin A, Wangkumhang P, Suwannasri P,
Ruangrit U, Agavatpanitch G, Tongsima S.
Genome Institute, National Center for Genetic Engineering and
Biotechnology, Pathumthani, Thailand
BMC Genomics. 2009 Dec 3;10 Suppl 3:S4.
This software is freely available at http://www4a.biotec.or.th/rexprimer
BACKGROUND: Polymerase chain reaction (PCR) is very useful in many
areas of molecular biology research. It is commonly observed that PCR
success is critically dependent on design of an effective primer pair.
Current tools for primer design do not adequately address the problem
of PCR failure due to mis-priming on target-related sequences and
structural variations in the genome.
METHODS: We have developed an integrated graphical web-based
application for primer design, called RExPrimer, which was written in
Python language. The software uses Primer3 as the primer designing core
algorithm. Locally stored sequence information and genomic variant
information were hosted on MySQLv5.0 and were incorporated into
RExPrimer.
RESULTS: RExPrimer provides many functionalities for improved PCR
primer design. Several databases, namely annotated human SNP databases,
insertion/deletion (indel) polymorphisms database, pseudogene database,
and structural genomic variation databases were integrated into
RExPrimer, enabling an effective without-leaving-the-website validation
of the resulting primers. By incorporating these databases, the primers
reported by RExPrimer avoid mis-priming to related sequences (e.g.
pseudogene, segmental duplication) as well as possible PCR failure
because of structural polymorphisms (SNP, indel, and copy number
variation (CNV)). To prevent mismatching caused by unexpected SNPs in
the designed primers, in particular the 3' end (SNP-in-Primer), several
SNP databases covering the broad range of population-specific SNP
information are utilized to report SNPs present in the primer
sequences. Population-specific SNP information also helps customize
primer design for a specific population. Furthermore, RExPrimer offers
a graphical user-friendly interface through the use of scalable vector
graphic image that intuitively presents resulting primers along with
the corresponding gene structure. In this study, we demonstrated the
program effectiveness in successfully generating primers for strong
homologous sequences.
CONCLUSION: The improvements for primer design incorporated into
RExPrimer were demonstrated to be effective in designing primers for
challenging PCR experiments. Integration of SNP and structural
variation databases allows for robust primer design for a variety of
PCR applications, irrespective of the sequence complexity in the region
of interest. This software is freely available at http://www4a.biotec.or.th/rexprimer
- BatchPrimer3: a high throughput web
application for PCR and sequencing primer design
You FM, Huo N, Gu YQ, Luo MC, Ma Y, Hane D, Lazo GR, Dvorak J, Anderson
OD.
Department of Plant Sciences, University of California, CA 95616, USA
BMC Bioinformatics. 2008 May 29;9:253.
BACKGROUND: Microsatellite (simple sequence repeat - SSR) and single
nucleotide polymorphism (SNP) markers are two types of important
genetic markers useful in genetic mapping and genotyping. Often,
large-scale genomic research projects require high-throughput
computer-assisted primer design. Numerous such web-based or
standard-alone programs for PCR primer design are available but vary in
quality and functionality. In particular, most programs lack batch
primer design capability. Such a high-throughput software tool for
designing SSR flanking primers and SNP genotyping primers is
increasingly demanded.
RESULTS: A new web primer design program, BatchPrimer3, is developed
based on Primer3. BatchPrimer3 adopted the Primer3 core program as a
major primer design engine to choose the best primer pairs. A new
score-based primer picking module is incorporated into BatchPrimer3 and
used to pick position-restricted primers. BatchPrimer3 v1.0 implements
several types of primer designs including generic primers, SSR primers
together with SSR detection, and SNP genotyping primers (including
single-base extension primers, allele-specific primers, and
tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing
primers. DNA sequences in FASTA format can be batch read into the
program. The basic information of input sequences, as a reference of
parameter setting of primer design, can be obtained by pre-analysis of
sequences. The input sequences can be pre-processed and masked to
exclude and/or include specific regions, or set targets for different
primer design purposes as in Primer3Web and primer3Plus. A
tab-delimited or Excel-formatted primer output also greatly facilitates
the subsequent primer-ordering process. Thousands of primers, including
wheat conserved intron-flanking primers, wheat genome-specific SNP
genotyping primers, and Brachypodium SSR flanking primers in several
genome projects have been designed using the program and validated in
several laboratories.
CONCLUSION: BatchPrimer3 is a comprehensive web primer design program
to develop different types of primers in a high-throughput manner.
Additional methods of primer design can be easily integrated into
future versions of BatchPrimer3. The program with source code and
thousands of PCR and sequencing primers designed for wheat and
Brachypodium are accessible at http://wheat.pw.usda.gov/demos/BatchPrimer3/
- Java web tools for PCR, in silico PCR, and
oligonucleotide assembly and analysis
Kalendar R, Lee D, Schulman AH.
MTT/BI Plant Genomics Laboratory, Institute of Biotechnology,
University of Helsinki, P.O. Box 65, FIN-00014 Helsinki, Finland
Genomics. 2011 Aug;98(2):137-144
The polymerase chain
reaction is fundamental to molecular biology and is the most important
practical molecular technique for the research laboratory. We have
developed and tested efficient tools for PCR primer and probe design,
which also predict oligonucleotide properties based on experimental
studies of PCR efficiency. The tools provide comprehensive facilities
for designing primers for most PCR applications and their combinations,
including standard, multiplex, long-distance, inverse, real-time,
unique, group-specific, bisulphite modification assays,
Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme
to design oligonucleotide sets for long sequence assembly by ligase
chain reaction. The in silico PCR primer or probe search includes
comprehensive analyses of individual primers and primer pairs. It
calculates the melting temperature for standard and degenerate
oligonucleotides including LNA and other modifications, provides
analyses for a set of primers with prediction of oligonucleotide
properties, dimer and G-quadruplex detection, linguistic complexity,
and provides a dilution and resuspension calculator.
- ConservedPrimers 2.0: a high-throughput
pipeline for comparative genome referenced intron-flanking PCR primer
design and its application in wheat SNP discovery
You FM, Huo N, Gu YQ, Lazo GR, Dvorak J, Anderson OD.
Department of Plant Sciences, University of California, Davis, CA
95616, USA
BMC Bioinformatics. 2009 Oct 13;10:331.
BACKGROUND: In some genomic applications it is necessary to design
large numbers of PCR primers in exons flanking one or several introns
on the basis of orthologous gene sequences in related species. The
primer pairs designed by this target gene approach are called
"intron-flanking primers" or because they are located in exonic
sequences which are usually conserved between related species,
"conserved primers". They are useful for large-scale single nucleotide
polymorphism (SNP) discovery and marker development, especially in
species, such as wheat, for which a large number of ESTs are available
but for which genome sequences and intron/exon boundaries are not
available. To date, no suitable high-throughput tool is available for
this purpose.
RESULTS: We have developed, the ConservedPrimers 2.0 pipeline, for
designing intron-flanking primers for large-scale SNP discovery and
marker development, and demonstrated its utility in wheat. This tool
uses non-redundant wheat EST sequences, such as wheat contigs and
singleton ESTs, and related genomic sequences, such as those of rice,
as inputs. It aligns the ESTs to the genomic sequences to identify
unique colinear exon blocks and predicts intron lengths.
Intron-flanking primers are then designed based on the intron/exon
information using the Primer3 core program or BatchPrimer3. Finally, a
tab-delimited file containing intron-flanking primer pair sequences and
their primer properties is generated for primer ordering and their PCR
applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of
the 6,045 in total) were found to have unique colinear exon blocks
suitable for primer design and 1,821 primer pairs were designed from
these single- or low-copy genes for PCR amplification and SNP
discovery. With these primers and subsequently designed genome-specific
primers, a total of 1,527 loci were found to contain one or more
genome-specific SNPs.
CONCLUSION: The ConservedPrimers 2.0 pipeline for designing
intron-flanking primers was developed and its utility demonstrated. The
tool can be used for SNP discovery, genetic variation assays and marker
development for any target genome that has abundant ESTs and a related
reference genome that has been fully sequenced. The ConservedPrimers
2.0 pipeline has been implemented as a command-line tool as well as a
web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/
- PriSM: a primer selection and matching tool
for amplification and sequencing of viral genomes
Yu Q, Ryan EM, Allen TM, Birren BW, Henn MR, Lennon NJ.
Broad Institute of MIT & Harvard, Cambridge, MA 02142, USA.
Bioinformatics. 2011 Jan 15;27(2):266-7. Epub 2010 Nov 9.
AVAILABILITY: The program is freely available for use at: http://www.broadinstitute.org/perl/seq/specialprojects/primerDesign.cgi
PriSM is a set of algorithms designed to select and match degenerate
primer pairs for the amplification of viral genomes. The design of
panels of hundreds of primer pairs takes just hours using this program,
compared with days using a manual approach. PriSM allows for rapid in
silico optimization of primers for downstream applications such as
sequencing. As a validation, PriSM was used to create an amplification
primer panel for human immunodeficiency virus (HIV) Clade B.
- PCRTiler: automated design of tiled and
specific PCR primer pairs
Gervais AL, Marques M, Gaudreau L.
Département de Biologie, Université de Sherbrooke, 2500
boul. de l'Université, Sherbrooke, Qc, J1K 2R1, Canada
Nucleic Acids Res. 2010 Jul;38(Web Server issue): W308-312
Efficiency and specificity of PCR amplification is dependent on several
parameters, such as amplicon length, as well as hybridization
specificity and melting temperature of primer oligonucleotides. Primer
design is thus of critical importance for the success of PCR
experiments, but can be a time-consuming and repetitive task, for
example when large genomic regions are to be scanned for the presence
of a protein of interest by chromatin immunoprecipitation experiments.
We present here a webserver that allows the automated design of tiled
primer pairs for any number of genomic loci. PCRTiler splits the target
DNA sequences into smaller regions, and identifies candidate primers
for each sub-region by running the well-known program Primer3 followed
by the elimination of primers with a high cross-hybridization potential
via BLAST. Tiling density and primer characteristics are specified by
the user via a simple and user-friendly interface. The webserver can be
accessed at http://pcrtiler.alaingervais.org:8080/PCRTiler
Additionally, users may download a standalone Java-based
implementation of this software. Experimental validation of PCRTiler
has demonstrated that it produces correct results. We have tiled a
region of the human genome, in which 96 of 123 primer pairs worked in
the first attempt, and 105 of 123 (85%) could be made to work by
optimizing the conditions of the PCR assay.
- maxAlike: maximum likelihood-based sequence
reconstruction with application to improved primer design for unknown
sequences
Menzel P, Stadler PF, Gorodkin J.
Center for non-coding RNA in Technology and Health, IBHV, University of
Copenhagen, Grønnegårdsvej 3, DK-1870 Frederiksberg,
Denmark.
Bioinformatics. 2011 Feb 1;27(3):317-25. Epub 2010 Dec 1.
AVAILABILITY: maxAlike is available for download and web server at: http://rth.dk/resources/maxAlike
MOTIVATION: The task of reconstructing a genomic sequence from a
particular species is gaining more and more importance in the light of
the rapid development of high-throughput sequencing technologies and
their limitations. Applications include not only compensation for
missing data in unsequenced genomic regions and the design of
oligonucleotide primers for target genes in species with lacking
sequence information but also the preparation of customized queries for
homology searches.
RESULTS: We introduce the maxAlike algorithm, which reconstructs a
genomic sequence for a specific taxon based on sequence homologs in
other species. The input is a multiple sequence alignment and a
phylogenetic tree that also contains the target species. For this
target species, the algorithm computes nucleotide probabilities at each
sequence position. Consensus sequences are then reconstructed based on
a certain confidence level. For 37 out of 44 target species in a test
dataset, we obtain a significant increase of the reconstruction
accuracy compared to both the consensus sequence from the alignment and
the sequence of the nearest phylogenetic neighbor. When considering
only nucleotides above a confidence limit, maxAlike is significantly
better (up to 10%) in all 44 species. The improved sequence
reconstruction also leads to an increase of the quality of PCR primer
design for yet unsequenced genes: the differences between the expected
T(m) and real T(m) of the primer-template duplex can be reduced by ~26%
compared with other reconstruction approaches. We also show that the
prediction accuracy is robust to common distortions of the input trees.
The prediction accuracy drops by only 1% on average across all species
for 77% of trees derived from random genomic loci in a test dataset.
- PrimerIdent: A web based tool for conserved
primer design
Pessoa AM, Pereira S, Teixeira J.
Bioinformation. 2010 Jul 6;5(2): 52-54
AVAILABILITY: http://primerident.up.pt
Conserved primers
across multiple species and simultaneously specific for a certain
isozyme can be rare and difficult to find. PrimerIdent was developed
aiming to automate this primer design and selection process in a given
nucleotide sequence alignment, providing an intuitive, easy to
interpret graphical result, which offers a list of all possible primers
that meet the user criteria, with a colour-code identity to each
sequence in the alignment. The software here presented is a simple and
intuitive web based tool that is suitable for distinguishing very
similar nucleotide sequences, such as isozymes-coding sequences, to
enable the conserved primer design across multiple species, necessary
for approaches that rely on knowing if a primer is suitable for a
certain set of pre-aligned sequences, to design a specific primer to a
certain sequence variation, or a combination thereof. This extremely
useful software can, therefore, be used as a tool for the specific
amplification of individual members of multigenic families across
related species and also to evaluate the differential expression of
isogenes for a given species.
- ThermoPhyl: a software tool for selecting
phylogenetically optimized conventional and quantitative-PCR
taxon-targeted assays for use with complex samples
Oakley BB, Dowd SE, Purdy KJ.
University of Warwick, School of Life Sciences, Coventry, UK
FEMS Microbiol Ecol. 2011 Jul;77(1): 17-27
The ability to specifically and sensitively target genotypes of
interest is critical for the success of many PCR-based analyses of
environmental or clinical samples that contain multiple templates.
Next-generation sequence data clearly show that such samples can
harbour hundreds to thousands of operational taxonomic units, a
richness that precludes the manual evaluation of candidate assay
specificity and sensitivity using multiple sequence alignments. To
solve this problem, we have developed and validated a free software
tool that automates the identification of PCR assays targeting specific
genotypes in complex samples. ThermoPhyl uses user-defined target and
nontarget sequence databases to assess the phylogenetic sensitivity and
specificity of thermodynamically optimized candidate assays derived
from primer design software packages. ThermoPhyl derives its name from
its central premise of testing Thermodynamically optimal assays for
Phylogenetic specificity and sensitivity and can be used for two primer
(traditional PCR) or two primers with an internal probe (e.g.
TaqMan(®) qPCR) application and potentially for oligonucleotide
probes. Here, we describe the use of ThermoPhyl for traditional PCR and
qPCR assays. PCR assays selected using ThermoPhyl were validated using
454 pyrosequencing of a traditional specific PCR assay and with a set
of four genotype-specific qPCR assays applied to estuarine sediment
samples.
- WASP: a Web-based Allele-Specific PCR assay
designing tool for detecting SNPs and mutations
Wangkumhang P, Chaichoompu K, Ngamphiw C, Ruangrit U, Chanprasert J,
Assawamakin A, Tongsima S.
Biostatistics and Informatics Laboratory, Genomics Institute, National
Center for Genetic Engineering and Biotechnology, Thailand Science
Park, Pathumtani, Thailand
BMC Genomics. 2007 Aug 14;8:275.
BACKGROUND: Allele-specific (AS) Polymerase Chain Reaction is a
convenient and inexpensive method for genotyping Single Nucleotide
Polymorphisms (SNPs) and mutations. It is applied in many recent
studies including population genetics, molecular genetics and
pharmacogenomics. Using known AS primer design tools to create primers
leads to cumbersome process to inexperience users since information
about SNP/mutation must be acquired from public databases prior to the
design. Furthermore, most of these tools do not offer the mismatch
enhancement to designed primers. The available web applications do not
provide user-friendly graphical input interface and intuitive
visualization of their primer results.
RESULTS: This work presents a web-based AS primer design application
called WASP. This tool can efficiently design AS primers for human SNPs
as well as mutations. To assist scientists with collecting necessary
information about target polymorphisms, this tool provides a local SNP
database containing over 10 million SNPs of various populations from
public domain databases, namely NCBI dbSNP, HapMap and JSNP
respectively. This database is tightly integrated with the tool so that
users can perform the design for existing SNPs without going off the
site. To guarantee specificity of AS primers, the proposed system
incorporates a primer specificity enhancement technique widely used in
experiment protocol. In particular, WASP makes use of different
destabilizing effects by introducing one deliberate 'mismatch' at the
penultimate (second to last of the 3'-end) base of AS primers to
improve the resulting AS primers. Furthermore, WASP offers graphical
user interface through scalable vector graphic (SVG) draw that allow
users to select SNPs and graphically visualize designed primers and
their conditions.
CONCLUSION: WASP offers a tool for designing AS primers for both SNPs
and mutations. By integrating the database for known SNPs (using gene
ID or rs number), this tool facilitates the awkward process of getting
flanking sequences and other related information from public SNP
databases. It takes into account the underlying destabilizing effect to
ensure the effectiveness of designed primers. With user-friendly SVG
interface, WASP intuitively presents resulting designed primers, which
assist users to export or to make further adjustment to the design.
This software can be freely accessed at http://bioinfo.biotec.or.th/WASP
About
QuantPrime - http://www.quantprime.de
QuantPrime is an
intuitive and user-friendly, fully
automated tool for primer pair design in small- to large-scale
real-time reverse transcription qPCR (also known as realtime qRT-PCR or
RT-qPCR) analyses. QuantPrime can be used on the website or on a local
computer (contact us for getting a copy); it offers design and
specificity checking with highly customizable parameters and is ready
to use with most publicly available eukaryotic transcriptomes,
including all higher eukaryote model organisms and important plant
crops, while benefiting from exon-intron border and splice variant
information in available genome annotations. Experimental results with
the model plant Arabidopsis thaliana, the crop Hordeum vulgare (barley)
and the model green alga Chlamydomonas reinhardtii show success rates
of designed primer pairs exceeding 96 %. For more
information on the algorithms used in QuantPrime, please read the paper
published in BMC Bioinformatics: QuantPrime - a flexible tool for
reliable high-throughput primer design for quantitative PCR.
The QuantPrime service
was created and is being maintained
by Samuel Arvidsson (supported by EU contract MRTN-CT-2006-035833) at
the University of Potsdam. The graphical design on the web site was
created by Dr. Mirosław Kwaśniewski, who also helped out with the
design of the program. The server is administrated by Diego Mauricio
Riaño-Pachón, who helped out with the design of the
program. The work is supervised by Prof. Dr. Bernd
Müller-Röber.
We kindly ask QuantPrime users for citation when primers
are used in published works. Please cite as follows:
QuantPrime - a flexible tool for reliable
high-throughput primer design for quantitative PCR.
Arvidsson S, Kwasniewski M, Riaño-Pachón DM,
Mueller-Roeber B.
Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm,
Germany
BMC Bioinformatics. 2008 9: 465
BACKGROUND: Medium- to
large-scale expression
profiling using quantitative polymerase chain reaction (qPCR) assays
are becoming increasingly important in genomics research. A major
bottleneck in experiment preparation is the design of specific primer
pairs, where researchers have to make several informed choices, often
outside their area of expertise. Using currently available primer
design tools, several interactive decisions have to be made, resulting
in lengthy design processes with varying qualities of the assays.
RESULTS: Here we present
QuantPrime, an intuitive and user-friendly,
fully automated tool for primer pair design in small- to large-scale
qPCR analyses. QuantPrime can be used online through the internet
http://www.quantprime.de/ or on a local computer after download;
it offers design and specificity checking with highly
customizable parameters and is ready to use with many publicly
available transcriptomes of important higher eukaryotic model organisms
and plant crops (currently
295 species in total), while benefiting from exon-intron border and
alternative splice variant information in available genome annotations.
Experimental results with the model plant Arabidopsis thaliana, the
crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii
show success rates of designed primer pairs exceeding 96%.
CONCLUSION: QuantPrime
constitutes a flexible, fully automated web
application for reliable primer design for use in larger qPCR
experiments, as proven by experimental data. 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. Future suggestions made by users can be easily
implemented, thus allowing QuantPrime to be developed into a
broad-range platform for the design of RNA expression assays.
Human
Endogenous
Control Gene Panel
For
all gene expression studies using quantitative PCR it is necessary to
compensate for differences between samples due to material losses,
differences in RT yields and PCR inhibition. Normalization should
include an endogenous control gene, but can also be complemented by
identical sample input amounts. The endogenous control gene should have
constant expression in all the samples compared. There is no universal
control gene, expressed at a constant level under all conditions and in
all tissues.
The best way to choose the proper reference gene is by running a panel
of potential genes on a number of representative test samples. The
gene(s) most appropriate for normalization are chosen in each case.
The Human Endogenous Control Panel consists of 12 validated
qPCR assays for the most common endogenous control genes
for gene expression studies, and provides a rapid and cost efficient
way to identify your control genes. The panel is compatible with most
commercial mastermixes containg SYBR Green I
=> short
manual
Polymerase Chain Reaction
is widely held as one of the most important inventions of the 20th
century in molecular biology. Small amounts of the genetic material can
now be amplified to be able to a identify, manipulate DNA, detect
infectious organisms, including the viruses that cause AIDS, hepatitis,
tuberculosis, detect genetic variations, including mutations, in human
genes and numerous other tasks.
PCR involves the following
three steps: denaturation, annealing and extension. First, the genetic
material is denatured, converting the double stranded DNA molecules to
single strands. The primers are then annealed to the complementary
regions of the single stranded molecules. In the third step, they are
extended by the action of the DNA polymerase. All these steps are
temperature sensitive and the common choice of temperatures is 94oC,
60oC and 70oC respectively. Good primer design is essential for
successful reactions. The important design considerations described
below are a key to specific amplification with high yield. The
preferred values indicated are built into all our products by default.
1. Primer Length: It is generally
accepted that the optimal length of PCR primers is 18-22 bp. This
length is long enough for adequate specificity, and short enough for
primers to bind easily to the template at the annealing temperature.
2. Primer Melting Temperature: Primer
Melting Temperature (Tm) by definition is the temperature at which one
half of the DNA duplex will dissociate to become single stranded and
indicates the duplex stability. Primers with melting temperatures in
the range of 52-58 oC generally produce the best results. Primers with
melting temperatures above 65oC have a tendency for secondary
annealing. The GC content of the sequence gives a fair indication of
the primer Tm. All our products calculate it using the nearest neighbor
thermodynamic theory, accepted as a much superior method for estimating
it, which is considered the most recent and best available.
Formula for primer Tm calculation:
Melting
Temperature Tm(oK) = {ΔH/ ΔS + R ln(C)},
Or Melting Temperature Tm(oC)
=
{ΔH/ ΔS + R ln(C)} - 273.15 where
ΔH (kcal/mole) : H is the
Enthalpy. Enthalpy is the amount of heat energy possessed by
substances. ΔH is the change in Enthalpy. In the above formula the ΔH
is obtained by adding up all the di-nucleotide pairs enthalpy values of
each nearest neighbor base pair.
ΔS (kcal/mole) : S is the
amount of disorder a system exhibits is called entropy. ΔS is change in
Entropy. Here it is obtained by adding up all the di-nucleotide pairs
entropy values of each nearest neighbor base pair. An additional salt
correction is added as the Nearest Neighbor parameters were obtained
from DNA melting studies conducted in 1M Na+ buffer and this is the
default condition used for all calculations.
ΔS (salt correction) = ΔS
(1M NaCl )+ 0.368 x N x ln([Na+])
Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.
[Na+] calculation:
[Na+] = Monovalent ion
concentration +4 x free Mg2+
3.Primer annealing
temperature : The primer melting temperature is the estimate
of the DNA-DNA hybrid stability and critical in determining the
annealing temperature. Too high Ta will produce insufficient
primer-template hybridization resulting in low PCR product yield. Too
low Ta may possibly lead to non-specific products caused by
a high number of base pair mismatches,. Mismatch tolerance is found to
have the strongest influence on PCR specificity.
Ta
= 0.3 x Tm(primer)
+ 0.7 Tm (product) – 14.9
where,
Tm(primer)
= Melting Temperature of the primers
Tm(product)
=
Melting temperature of the product
4. GC Content :
The GC content (the number of G's and C's in the primer as a percentage
of the total bases) of primer should be 40-60%.
5. GC Clamp :
The presence of G or C bases within the lat five bases from the 3' end
of primers (GC clamp) helps promote specific binding at the 3' end due
to the stronger bonding of G and C bases. More than 3 G's or C's should
be avoided in the last 5 bases at the 3' end of the primer.
6. Primer Secondary
Structures : Presence of the primer secondary structures
produced by intermolecular or intramolecular interactions can lead to
poor or no yield of the product. They adversely affect primer template
annealing and thus the amplification. They greatly reduce the
availability of primers to the reaction.
i) Hairpins : It is formed
by intramolecular interaction within the primer and should be avoided.
Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal
hairpin with a ΔG of -3 kcal/mol is tolerated generally.
ΔG definition : The Gibbs
Free Energy G is the measure of the amount of work that can be
extracted from a process operating at a constant pressure. It is the
measure of the spontaneity of the reaction. The stability of hairpin is
commonly represented by its ΔG value, the energy required to break the
secondary structure. Larger negative value for ΔG indicates stable,
undesirable hairpins. Presence of hairpins at the 3' end most adversely
affects the reaction.
ΔG = ΔH – TΔS
ii) Self Dimer : A primer
self-dimer is formed by intermolecular interactions between the two
(same sense) primers, where the primer is homologous to itself.
Generally a large amount of primers are used in PCR compared to the
amount of target gene. When primers form intermolecular dimers much
more readily than hybridizing to target DNA, they reduce the product
yield. Optimally a 3' end self dimer with a ΔG of -5 kcal/mol and an
internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.
iii) Cross Dimer : Primer
cross dimers are formed by
intermolecular interaction between sense and antisense primers, where
they are homologous. Optimally a 3' end cross dimer with a ΔG of -5
kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is
tolerated generally.
7. Repeats : A
repeat is a di-nucleotide occurring many times consecutively and should
be avoided because they can misprime. For example: ATATATAT. A maximum
number of di-nucleotide repeats acceptable in an oligo is 4
di-nucleotides.
8. Runs :
Primers with long runs of a single base should generally be avoided as
they can misprime. For example, AGCGGGGGATGGGG has runs of base 'G' of
value 5 and 4. A maximum number of runs accepted is 4bp.
9. 3' End Stability :
It is the maximum ΔG value of the five bases from the 3' end. An
unstable 3' end (less negative ΔG) will result in less false priming.
10. Avoid Template
secondary structure : A single stranded Nucleic acid sequences
is highly unstable and fold into conformations (secondary structures).
The stability of these template secondary structures depends largely on
their free energy and melting temperatures(Tm).
Consideration of template secondary structures is important in
designing primers, especially in qPCR. If primers are designed on a
secondary structures which is stable even above the annealing
temperatures, the primers are unable to bind to the template and the
yield of PCR product is significantly affected. Hence, it is important
to design primers in the regions of the templates that do not form
stable secondary structures during the PCR reaction. Our products
determine the secondary structures of the template using the Mfold
algorithm and design primers avoiding them.
11. Avoid Cross
homology :
To improve specificity of the primers it is necessary to avoid regions
of homology. Primers designed for a sequence must not amplify other
genes in the mixture. Commonly, primers are designed and then BLASTed
to test the specificity. Our products offer a better alternative. You
can avoid regions of cross homology while designing primers. You can
BLAST the templates against the appropriate non-redundant database and
the software will interpret the results. It will identify regions
significant cross homologies in each template and avoid them during
primer search.
Parameters for
Primer Pair Design:
1. Amplicon Length :
The amplicon length is dictated by the experimental goals. For qPCR,
the target length is closer to 100 bp and for standard PCR, it is near
500 bp. If you know the positions of each primer with respect to the
template, the product is calculated as: Product length = (Position of
antisense primer-Position of sense primer) + 1.
2. Product position :
Primer can be located near the 5' end, the 3' end or any where within
specified length. Generally, the sequence close to the 3' end is known
with greater confidence and hence preferred most frequently.
3. Tm of Product :
Melting Temperature (Tm) is the temperature at which one
half of the DNA duplex will dissociate and become single stranded. The
stability of the primer-template DNA duplex can be measured by the
melting temperature (Tm).
4.Optimum Annealing
temperature (Ta Opt): The formula of Rychlik is
most respected. Our products use this formula to calculate it and
thousands of our customers have reported good results using it for the
annealing step of the PCR cycle. It usually results in good PCR product
yield with minimum false product production.
Ta
Opt = 0.3 x(Tm
of primer) + 0.7 x(Tm of product) - 25
where
Tm of primer is the melting temperature of the less stable
primer-template pair
Tm of product is the melting temperature of the PCR product.
5. Primer Pair Tm
Mismatch Calculation : The two primers of a primer pair should
have closely matched melting temperatures for maximizing PCR product
yield. The difference of 5oC or more can lead no
amplification.
Primer Design Using
Software
A
number of primer design tools are available that can assist in PCR
primer design for new and experienced users alike. These tools may
reduce the cost and time involved in experimentation by lowering the
chances of failed experimentation.
Primer
Premier
follows all the guidelines specified for PCR primer design. Primer
Premier can be used to design primers for single templates, alignments,
degenerate primer design, restriction enzyme analysis. contig analysis
and design of sequencing primers.
The guidelines for qPCR primer
design vary slightly. Software such as AlleleID
and Beacon
Designer
can design primers and oligonucleotide probes for complex detection
assays such as multiplex assays, cross species primer design, species
specific primer design and primer design to reduce the cost of
experimentation.
PrimerPlex
is a software that can design ASPE (Allele specific Primer Extension)
primers and capture probes for multiplex SNP genotyping using
suspension array systems such as Luminex xMAP® and BioRad Bioplex.
References
:
1. “A critical review of
PCR primer design algorithms and cross-hybridization case study” By
F.John Burpo.
2. “Optimization of the annealing temperature for DNA amplification in
vitro” By W.Rychlik, W.J.Spencer
and R.E.Rhoads.
3. “A unified view of polymer, dumbbell, and oligonucleotide DNA
nearest-neighbor thermodynamics” By John SantaLucia.
4. “A computer program for selection of oligonucleotide primers for
polymerase chain reactions” Lowe T, Sharefkin J, Yang SQ, Dieffenbach
CW.
5. “Optimization strategies for the polymerase chain reaction” Williams
JF.Perkin-Elmer Corporation, Norwalk, CT 06859-0251.
6. “Algorithms and thermodynamics for RNA secondary structure
prediction. A Practical guide.” Zuker.m.athews, D.Turner, D.
Summary
of Primer Design Softwares:
qPCR is our language -
Cq, r2, ΔRn, S:N, MIQE
are part of our everyday vocabulary
True qPCR platform
independence with BHQ® Probes
BHQ Probes
are compatible with most popular thermal cyclers
Fluorescent-quenched oligo, typically 20-30
bases in length
Designed to bind at 70 °C for qPCR thermal cycling
Participate in FRET and static quenching for high signal-to-noise
ratios and exquisite sensitivity
Perfect for gene expression analysis, copy number determination and SNP
genotyping
Free qPCR Assay
Design using RealTimeDesign™ Software
Need design help? Register an account to use RealTimeDesign Software,
Biosearch’s free qPCR probe and primer design program.
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Biosearch
Technologies
introduces advanced qPCR design software
Biosearch Technologies,
Inc.
(BTI), innovative developer of the patented Black Hole Quencher®,
CAL
Fluor®, Quasar® and Pulsar® series of quenchers and dyes
for
multiplex qPCR, recently released the first in a series of qPCR assay
probe
and primer design modules for its new web-based software engine,
RealTimeDesign™.
Hosted on
BTI's website, www.biosearchtech.com,
RealTimeDesign software is available free of charge and can be used on
any desktop computer having internet access. This first module for
TaqMan® assay design (TaqMan is a registered trademark of Roche
Molecular Systems, Alameda, CA) significantly refines and enhances
existing TaqMan-proven assay design algorithms with the latest insights
into rules governing primer-dimer formation, amplification efficiency,
secondary structure and mis-hybridizations.
Whether
the user is a novice or seasoned expert in assay design, RealTimeDesign
ensures that TaqMan assays will routinely demonstrate detection and
amplification efficiencies averaging 99%.
For the
novice user, RealTimeDesign™ Express Mode takes all the guess work out
of assay
design and fully automates all steps of the assay design process. For
more
experienced users, Custom Mode allows user-defined design parameter
modifications
at every step of the assay design process. In both Express and Custom
Mode,
assays can be designed against 1 to 10 different targets simultaneously
with
results archived for future review. Visit www.qPCRdesign.com
to learn more.
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Sigma
is pleased to offer OligoArchitect for all of your primer and probe
design requirements. OligoArchitect includes both our complimentary
online design tool and our unique consultative service.
For routine needs, improve your assay with our
OligoArchitect online
design tool powered by the industry standard Beacon Designer™ (Premier
Biosoft). The user-friendly interface utilizes the latest algorithms,
provides results in real time, supports templates up to 10,000 base
pairs, and allows for the adjustment of input parameters such as
homopolymer run/repeat maximum length, G/C clamp length, and maximum
primer pair Tm
mismatch.
Designs can be
completed for traditional PCR or
quantitative real-time PCR (qPCR) using the following detection
chemistries:
- SYBR®
Green I
- Dual-Labeled Probes
Our online design tool
can be used for the
following applications:
- Traditional PCR
- Endpoint genotyping
- Gene expression analysis
- Genomic copy number determination
- Allele discrimination
- SNP detection
- Haplotyping
OligoArchitect online is easy to use: select
either ‘SYBR Green’ or
‘Dual Labeled Probe’, type in the name of your assay, paste in your
sequence, and click search. You can add and delete a SNP or select
‘Primer Parameter’ if you wish to adjust the default settings. Download
the glossary (387 Kb PDF) to
learn more about the various parameters.
All reported sequences, associated properties,
and assay parameters
are available for export to Excel and convenient email ordering.
To establish MIQE-compliance,
be sure to
- check the designed sequences for cross
homology using Blastn and
- check for secondary structure formation in
the target using mFold
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Assay
Design Tools for qPCR
IDT offers a group of qPCR design tools to suit your specific
application needs. Whether you want to quickly order a predesigned
assay, or desire assistance with designing assays for a challenging
target, IDT has a design tool that can help. With three different
programs to choose from, it is important to know which will provide the
optimal qPCR assay for your needs.
Each of the three design programs requires only the NCBI RefSeq number,
which can be used to pull the target sequence from NCBI. If you are
working with unannotated sequences, the freely available RealTime and
PrimerQuestSM programs can also design assays for a user-specified
target.
Key Features of Assay Design Tools
PrimeTime® qPCR Program
- Optimal design for human, mouse, and rat mRNA
targets
- Incorporates bioinformatics accounting for
factors such as SNPs, BLAST searches to avoid cross-reaction and
off-target amplification, and recognition of splice variants
RealTime PCR Program
- Designs specifically for qPCR, with some
customization possible
- Optimal design for targets that are not human,
mouse, or rat
- Can be used to effectively design assays for
use with intercalating dyes such as SYBR® Green I (Molecular
Probes, Inc.)
PrimerQuestSM PCR Primer
Design
- Highly customizable option for difficult qPCR
designs
PrimerQuestSM
PrimerQuest is a useful tool for qPCR assay designs
with non-standard requirements. This program is not specific for qPCR
design like our other two programs, but if your design requires more
demanding customization, this highly flexible program can be of great
use. PrimerQuest designs can be customized in many ways, such as
directing the assay towards certain areas of your target or by
specifying primer or probe sequences. IDT Technical Support can also
offer assistance with this program to help you meet your specific
design challenges.
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PrimeSyn Lab
Prime Syn Lab provides custom
DNA synthesis services ranging from custom oligos and simple primers to
labeled
probes (FRET,Scorpion, probes for RTPCR). Other services include
protein
analysis, protein purification, and analytical method development.
http://www.primesyn.com
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PCR Primer
Design Guidelines
Polymerase Chain Reaction is widely held as one of the most important
inventions of the 20th century in molecular biology. Small amounts of
the genetic material can now be amplified to be able to a identify,
manipulate DNA, detect infectious organisms, including the viruses that
cause AIDS, hepatitis, tuberculosis, detect genetic variations,
including mutations, in human genes and numerous other tasks.
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
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PRaTo,
a web-tool for the selection
of the best primer pair for qPCR
http://prato.daapv.unipd.it
PRaTo: A web-tool to select optimal primer
pairs for qPCR
Alberto Nonis, Marco Scortegagna, Alessandro Nonis, Benedetto Ruperti
Biochemical and Biophysical Research Communications 415 (2011) 707–708
An essential pre-requisite to perform sound quantitative real-time
polymerase chain reaction (qPCR) assays is to design outstanding primer
pairs. This means they must have a good efficiency and be not prone to
produce multiple amplicons or primer dimer products. To circumvent
these issues, several softwares are available to help primer design.
Although satisfactory computer-aided primer design tools are available
for standard PCR, less efforts were done to provide specific methods
for selection of optimal primer pairs for qPCR. We have developed PRaTo
a web-based tool that enables checking and ranking of primers pairs for
their attitude to perform optimally and reliably when used in qPCR
experiments. PRaTo is available at http://prato.daapv.unipd.it
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Design
of Primers for
Automated Sequencing
One of the most important factors in successful automated DNA
sequencing is proper primer design. This document describes the steps
involved in this process and the major pitfalls to avoid.
****
Use a Computer to
Design Primers ****
We highly recommend that a computer be used during primer design in
order to check for certain fatal design flaws. Numerous programs are
capable of performing this analysis. For example, look for 'Primer3' on
the web.
Some Basic Concepts: If you are confused by the strands and primer
orientation, read this.
Sequencing primers must be able to anneal to the target DNA in a
predictable location and on a predictable strand. They furthermore must
be capable of extension by Taq DNA Polymerase.
Some people are confused about how to examine a DNA sequence to choose
an appropriate primer sequence. Here are a few things for novices to
remember:
- Sequences are always written from 5' to 3'.
This includes the sequence of your template DNA (if known), the
sequence of the vector DNA into which it is inserted, and the sequence
of proposed primers. Don't ever write a primer sequence reversed or you
will only confuse yourself and others.
- Polymerase always extends the 3' end of the
primer, and the sequence you will read will be the same strand (sense
or anti-sense) as the primer itself.
- Thus, if you choose a primer sequence that you
can read in your source sequence (for example, in the vector), the
sequence you will obtain will extend from the primer's right (3') end.
- Conversely, if you choose a primer from the
strand opposite to what your 'source' sequence reads, the resulting
sequence will read towards the left.
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Alkami Biosystems Quick
Guide for PCR
This 158 page PCR manual covers the following
topics: PCR Primer design, PCR Methods, PCR Polymerases, PCR Variables,
PCR Troubleshooting, Special PCR Topics, Appendixes and a comprehensive
Index. This FREE online guide is in PDF format.
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ExonPrimer is a Perl script that helps to design intronic primers for
the PCR amplification of exons. The script needs a cDNA and the
corresponding genomic sequence as input. It aligns these sequences
using Blat and designs PCR
primers to amplify each exon using
Primer3. The positions of the exons are deduced from the alignment
of the genomic and the cDNA sequences. Insertions/deletions up to 6
base pairs are bridged by postprocessing. Exons with small introns
in-between are combined. Exons smaller than 20-25 bp will not be
recognized. The user can define the
maximum exon size. Exons larger than this size will be divided into
several
parts.
The poly-A tail of the cDNA should be clipped to allow the alignment of
the cDNA and the genomic DNA sequence. The genomic sequence must be
longer than the cDNA sequence. Otherwise the design of primers for the
first and/or last exon is not possible.
Download
of the human genome sequence with all SNPs masked by N's. Using this
sequence, one can avoid primers to be positioned across SNPs.
ExonPrimer is also available in the
UCSC Genome Browser for the human genome assemblies hg16 (July
2003) and hg17 (May 2004). One can find a link to ExonPrimer in the
'Quick Links to Tools and Databases' section of the known genes details
page.
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Design of Primers for Automated Sequencing
A detailed guide for designing
primers for automated sequencing
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GeneFisher (manual pages)
Interactive Primer Design ( Folker
Meyer & Chris
Schleiermacher)
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