SNP Genotyping Assay Design
To successfully assay a SNP using FP-TDI we must design two PCR primers and at least one extension primer, also called a SNP primer.
Our PCR primers are synthesized oligos between 20 and 26 base pairs in length with a TM of around 55 degrees. Our SNP primers are typically 14 to 30
bases in length with a TM range of 50-55 degrees.
View our references.
When possible, we obtain our flanking sequence for primer design from allocation files created by the
International HapMap Project. These files contain a wealth of information about each SNP including observed alleles and 1,000 bp of NCBI-masked sequence for each flank.
Once flanking sequence is obtained, we perform assay design in several steps:
repeat masking of the flanking sequence,
PCR primer design using primer3,
SNP primer design in each orientation, and
SNP primer selection to get the best results.
Repeat Masking
We make two passes at PCR primer design, each with a different repeat masking system. First, we attempt probe design using NCBI's masking, denoted by lower case
flanking sequence in the allocation files. In the second pass we discard NCBI's masking and use Arian Smit's RepeatMasker with the latest libraries from RepBase. Should
both primer design attempts succeed, we use the primer set obtained using NCBI's masking.
PCR Primer Design
We use primer3, a freeware UNIX-based application, to design our PCR primers. This program takes a variety of input parameters and selects the best primer pair for an
amplification target while checking for primer-dimer, misincorporation, and other potential problems. We specify a PCR product size of 80-400 bp and a primer GC content of
20-50%. In addition, we configure primer3 so that neither PCR primer is within 30 bases of the SNP to ensure that no probe overlapping is possible.
SNP Primer Design
If a set of PCR primers was obtained, we next attempt design of up to two TDI primers, one for each orientation (called 'p3' for forward orientation, 'p4' for reverse
orientation). TDI primers are designed using unmasked DCC flanking sequence, since a PCR product of 80-400 bp contains the target for TDI primer hybridization.
The most "ideal" primer is found for each orientation, if possible, by starting at the position just upstream (5') of the SNP, and adding bases in the upstream (5')
direction until the minimum SNP primer length, 14 base pairs, is reached. At that point, we calculate the TM of the primer using Nearest Neighbor Theromodynamics
(see [1] and [2]). If the TM is between 50 and 55 degrees (inclusive), meets our requirements and can be used. Otherwise, bases are added one at a time in the
5' direction until either (1) a primer of sufficient TM is found, or (2) a primer length of 30 bases is reached, at which point the TDI primer design for this
orientation fails. If at any time an ambiguity code reveals the presence of a neighbor SNP in the TDI primer sequence, the TDI primer is automatically failed.
TDI primer design is always attempted for both orientations; for each orientation the shortest TDI primer that meets TM requirements is chosen.
SNP Primer Selection
Since TDI primer design uses unmasked flanking sequence, we perform additional analyses of each TDI primer to assess its quality. Primers are assigned a quality score
based upon the number of single-base and three-base repeats, as well as their size in bases. (Shorter is better, since oligo cost is directly associated with oligo
size). Some primers are automatically failed for the repeats they contain. Also taken into consideration is whether or not the last base of the SNP primer is the
same as one of the dye alleles - in their research with Texas Red, Ming Xiao et al found that such primers can interfered with dye incorporation (see [3]).
If only one orientation's primer passes all criteria to this point, it will be used for
the assay. Otherwise, the TDI with the best quality score is used first. Often, repeating an assay with the other orientation of TDI primer will rescue a failed
assay.
References
[1] Beasley, E.M., R.M. Myers et al. 1999. Statistical Refinement of Primer Design Parameters., pp. 55-71. In D.H.G. Michael A. Innis, John J. Sninsky
[2] Owczarzy, R., Vallone, P.M. et al. Predicting Sequence-Dependent Melting Stability of Short Duplex DNA Oligomers. Biopoly 44: 217-239.
[3] Xiao, M., Kwok, PY. et al. 2004. Role of Excess Inorganic Pyrophosphate in Primer-Extension Genotyping Assays. Genome Res. 14(9):1749-55.
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