Tutorial/help

How to use MRPrimerW

MRPrimerW

  • Why MRPrimerW?
  • MRPrimerW is available for free and open website for searching desired primers instantly while varying filtering constraints by users.

    MRPrimerW has three distinctive features:
    (i) searching top-1 primer pairs for multiple target gene(s) that satisfy the user-defined same stringent and allele-invariant constraints for qPCR at once.
    (ii) providing search modes: fast mode and exact mode.
    (iii) supporting several ways to search, such as by NCBI gene symbol, NCBI CCDS ID, NCBI gene ID, GenBank Accession number, GenBank aliases, or keyword (gene description).
    * See more details in About page.

Search: basic parameters

  • Species
  • Currently, MRPrimerW provides primers from human and mouse genes.

  • Search by
  • User may search primers by NCBI gene symbol(s), GenBank Accession number(s), NCBI CCDS ID(s), NCBI gene ID(s), Aliases, or keywords (gene description). User can find exact search terms from NCBI GenBank.

  • For text
  • User may search either a single gene or multiple genes.

    Examples (for single target gene, species: human)

  • NCBI gene symbol: ACTB
  • GenBank Accession number: NM_001101.3
  • NCBI CCDS ID: CCDS5341.1
  • NCBI gene ID: 60
  • Aliases: BRWS1
  • keywords (gene description): actin beta
  • TaqMan probe design mode
  • The user can select the TaqMan probe design option to design TaqMan probes as well as SYBR Green primers.
  • Email option
  • The user can enter his/her email address, and the web server sends an email containing a link to the result page to the user after query processing is completed. The result page accessible via the link in the email is available for two weeks (i.e., 14 days).

Search: advanced settings

  • Single filtering
    • Primer length: Minimum and maximum acceptable length of a primer. Must be greater than or equal to 17 and less than or equal to 27 (see About page).
    • Melting temperature(Tm): Minimum and maximum acceptable melting temperature(℃) for a primer. Must be greater than or equal to 56 and less than or equal to 64 (see About page). For calculating the melting temperature, we adopted the nearest-neighbor thermodynamic model (ref).
    • GC content: Minimum and maximum acceptable percentage of Gs and Cs in a primer. Must be greater than or equal to 30 and less than or equal to 70 (see About page).
    • Self-complementarity: Maximum acceptable number of primer bases annealing to itself. Must be less than maximum primer length.
    • 3' end self-complementarity: Maximum acceptable number of primer 3' end bases annealing to itself. Must be less than half of maximum primer length.
    • Contiguous residue: Maximum acceptable length of a mononucleotide repeat, for example AAAAAA. Must be less than 5 (see About page).
    • End stability(∆G, kcal/mol): Maximum stability for the last five 3' bases of primer. The stability is calculated using the nearest-neighbor thermodynamic model (ref).
  • Pair filtering
    • Length difference: Minimum and maximum acceptable difference between the lengths of forward and reverse primers.
    • Melting temperature(Tm) difference: Minimum and maximum acceptable difference between the melting temperatures of forward and reverse primers.
    • Product size: Minimum and maximum acceptable size of PCR product produced by reaction between forward and reverse primers.
    • Pair-complementarity: Maximum acceptable number of bases of forward primer to bind to the reverse primer, and vice versa.
    • 3' end pair-complementarity: Maximum acceptable number of bases of the 3' end forward primer to bind to the 3' end reverse primer, and vice versa.
  • Recommended settings (for qPCR)
Parameters Values
Single filtering primer length 19~23 bp
melting temperature (TM) 60~63℃
GC content 35~65%
self-complementarity < 5-mer
3’ self-complementarity < 4-mer
contiguous residue < 6-mer
End stability (∆G) >= -9 kcal/mol
Pair filtering length difference <= 3-mer
TM difference <= 5℃
product size 100~250 bp
pair-complementarity < 9-mer
3’ pair-complementarity < 4-mer

Analysis of result data

  • How to interpret result?
  • The first table shows top primer pairs for specific target genes. The second table shows a set of less target-specific primers for the query genes having no result in the first table. Both tables show the top-1 primer pair sequences, GenBank Accession number with link for the details of gene and information on the primer, such as penalty score (i.e. a lower penalty score indicates a better primer), amplicon size, Tm, and position.

    • ① No.: index
    • ② Gene: if search type is NCBI CCDS ID or NCBI Gene ID, then CCDS ID or Gene ID will be shown. For other search type (NCBI Gene Symbol, Aliases, or keyword), Gene symbol will be shown.
    • ③ Accession number: links to NCBI GenBank
    • ④ Penalty score: primer pairs with low scores have high rank.
    • ⑤ Forward primer: sequence of forward primer
    • ⑥ Reverse primer: sequence of reverse primer
    • ⑦ Forward TM: temperature of forward primer
    • ⑧ Reverse TM: temperature of reverse primer
    • ⑨ Amplicon size: size of product
    • ⑩ Forward position: position of forward primer
    • ⑪ Reverse position: position of reverse primer

    Also by default, the output table is sorted by Gene in ascending order (②). The user can sort the output table in various ways: forward position and reverse position in descending or ascending order (⑩ and ⑪).

  • If MRPrimerW cannot find any primers?
  • No primers due to too strict parameters
    the set of genes that may have target-specific primer pairs by relaxing filtering constraints and the guidelines how to adjust the constraints.

    Not found target gene(s)
    the set of genes that may be wrong or have typos.