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