Genotyping Assay Design for Single Base Extention and FP-TDI Detection

Description:
For each SNP two PCR primers and a forward and reverse single base extention SNP
primer design was attempted. After obtainign uniquely mapped SNPs with other known
SNPs marked in the sequence the design was attempted in two different steps: 1) SNP
primer design and 2) PCR primer design.

1) SNP Primer Design

The TM of the shortest possible (14 bases) was calculated using Nearest Neighbor
Theromodynamics (Beasley, E.M., R.M. Myers, D.R. Cox and L.C. Lazzeroni. 1999.
Statistical Refinement of Primer Design Parameters., pp. 55-71. In D.H.G. Michael A.
Innis, John J. Sninsky ). If the primer TM was less than 50 degrees C the primer was
extended another base until the TM was between 50-55 degrees (using Allawi and
SantaLucia Nearest Neighbor Sequence Dependent Thermodynamic Parameters as
described in Owczarzy, R., Vallone, P.M., Gallo, F.J., Paner, T.M., Lane, M.J. 1997.
Predicting Sequence-Dependent Melting Stability of Short Duplex DNA Oligomers.
Biopoly 44: 217-239.) or until the length of the primer as greater than 40 bases.
If a primer between 14-40 bases long with a TM of 50-55 degrees was found for
both the forward and reverse SNP primers a preferred SNP primer was chosen to try
exprimentally by assigning penalties according to the criteria in Table 1. The penalty
scores are then compared. If the primers have different penalty scores pick the one
with the lowest. If the penalty scores are the same and the alleles are C/T or G/A pick
the primer that will result in genotyping C/T (best for FP-TDI), other wise pick the shortest
primer (cheapest). If a primer is still not picked default to picking forward primer.

2) PCR Primer Design

    Following Repeat Masking of sequence, PCR Primer were designed using Primer3 release 0.9
    (Steve Rozen, Helen J. Skaletsky (1996,1997,1998) Primer3. Code available at
    http://www-genome.wi.mit.edu/genome_software/other/primer3.html) with similar parameters
    as described previously (Vieux, E.F., Kwok, P.Y., Miller, R.D.Primer design for PCR and sequencing
    in high-throughput analysis of SNPs.Biotechniques32:S28-S32), with the minor changes:
        TARGET= SNP_Positon-20 bases, 20 bases
        PRIMER_OPT_SIZE=23
        PRIMER_MAX_SIZE=26
        PRIMER_MIN_SIZE=20
        PRIMER_OPT_TM=55
        PRIMER_MAX_TM=56
        PRIMER_MIN_TM=54
        PRIMER_PRODUCT_SIZE_RANGE=80-300
        PRIMER_PRODUCT_OPT_SIZE=250
        PRIMER_PAIR_WT_PRODUCT_SIZE_LT=.20
        PRIMER_PAIR_WT_PRODUCT_SIZE_GT=.20
        PRIMER_MIN_GC=20
        PRIMER_MAX_GC=50
        PRIMER_SALT_CONC=50
        PRIMER_SELF_ANY=8
        PRIMER_SELF_END=3
        PRIMER_DNA_CONC=40
        PRIMER_GC_CLAMP=0
        PRIMER_MAX_END_STABILITY=8
        PRIMER_NUM_RETURN=1