1) A set of blocks is input, where a block is an aligned array of amino acid
sequence segments without gaps that represents a highly conserved region of
homologous proteins. A weight is provided for each sequence segment, which can be
increased to favor the contribution of selected sequences in designing the primer.
A codon usage table is chosen for the target genome.
2) An amino acid position-specific scoring matrix (PSSM) is computed for each block
using the odds ratio method.
3) A consensus amino acid residue is selected for each position of the block as the
highest scoring amino acid in the matrix.
4) For each position of the block, the most common codon corresponding to the amino
acid chosen in step 3 is selected utilizing the user-selected codon usage table.
This selection is used for the default 5' consensus clamp in step 8.
5) A DNA PSSM is calculated from the amino acid matrix (step 2), genetic code
table and codon usage table. The DNA matrix has three positions for each
position of the amino acid matrix. The score for each amino acid is divided among
its codons in proportion to their relative weights from the codon usage table, and
the scores for each of the four different nucleotides are combined in each DNA
matrix position. Nucleotide positions are treated independently when the scores are
combined. As an option, the highest scoring nucleotide residue from each position
can replace the most common codons from step 4 that are used in the consensus clamp.
6) The degeneracy is determined at each position of the DNA matrix based on the
number of bases found there. As an option, a weight threshold can be specified such
that bases that contribute less than a minumum weight are ignored in determining
degeneracy.
7) Possible degenerate core regions are identified by scanning the DNA matrix in
the 3' to 5' direction. A core region must start on an invariant 3' nucleotide
position, have length of 11 or 12 positions ending on a codon boundary, and have a
maximum degeneracy of 128 (current default). The degeneracy of a region is the
product of the number of possible bases in each position.
8) Candidate degenerate core regions are extended by addition of a 5' consensus clamp
from step 4 or 5. The length of the clamp is controlled by a melting point temperature
calculation (current default = 60o) and is usually ~20 nucleotides.
9) Steps 7 and 8 are repeated on the reverse complement of the DNA matrix from step
5 for primers corresponding to the opposite DNA strand.
