PCR Primers: Conserved Primer Design for Wheat SNP
Discovery
Objectives:
Development of conserved PCR primers

Materials:
EST sequences:
Four sets of EST sequences were selected for primer design:
  1. Mapped contigs and singletons: The total of 4186 contigs (unigenes) and 1859 singletons
    mapped to wheat chromosomes were obtained by a query from the wEST databases ( in
    GrainGene database ).
  2. Mapped single 5’ EST sequences: a total of 6426 5' EST sequences were obtained from wEST
    database.
  3. Mapped single 3’ EST sequences: a total of 5814 3' EST sequences were obtained from wEST
    database.
Rice sequences:
A total of 4074 rice sequences with greater than 10kb and less than 1Mbp were downloaded from
RiceGAAS(http://ricegaas.dna.affrc.go.jp/). All sequences have 545,430,227 bp long.

Pipeline of Conserved PCR primer design:
  • The pipeline of conserved primer design is illustrated in Figure 1.        
  • Blastn search against rice sequence database:
    Parameters: -b 10 –W 15 –E 3 –G 1 –U –e 1e10
  • Intron/exon analysis (alignment of introns/exons):
  • Find all matches with bit score > 50 or expect value > 1e-10. Each match corresponds to
    an exon in wheat EST or rice squence.
  • Exclude the exon(s) from ESTs in which the same slice of sequence has more than one
    matches (repeative elements)
  • Exclude the EST contigs in which slices of sequences have wrong order of matches
    against rice sequences. This is probably due to the incorrect EST assembly.
  • Exclude the ESTs with no exon or only one exon found from blastn search.
  • Calculate the length of exons and introns, and find the coordinates of each exon and intron.
  • Create a new sequence for each EST or contig, in which the intron sequences were
    inserted between two exons, but intron sequences were replaced by “N”, e.g., “….
    GATCGGTTTACNNNNNNNNN….NNNNGGTTCAATT….”. This new sequence was used to
    design primer in Primer3 program. The advantages to use this new sequence rather than
    the original EST sequence are that (1) primers can surely be picked only from exon
    regions, and (2) the product size, and number and length of introns and exons included in
    product can be estimated, (3) in primer analysis, we can check if the primers picked using
    Primer3 come from different exons or from the same exon region.
  • Primer design:
                          Parameters used in Primer3 program:
Parameters
 Min
 Optimum
 Max
Product size
 400
 800
 1500
Tm
 55
 60
 65
Primer length
 18
 20
 25
GC content
 30
   70
             Note: only one primer set was picked for each EST or contig.

  • Primer analysis:
  • Assign the corresponding EST sequence name, accession number, contig name (if any),
    chromosome position, number of match exons, coordinates of exons in sequences, match
    score of each exon, etc. to each primer set.
  • Find out the exons the primer sequences were picked from. If the primers are picked from
    the same exon, the primer set will be removed and re-picked manually later.
  • Estimate the product size, number of introns and exons included in the amplifying product,
    length of introns and exons included in the amplifying product.         
  • Transfer all related information into databases.
Several Java and Perl programs have been developed. Those programs along with blastn and primer3
programs, as well as rice sequence database compose a pipeline of the conserved primer design