SNP Design
References
A few references I used to give me ideas on how to design primers for the SNPs are:
Drenkard E, Richter BG, Rozen S, Stutius LM, Angell NA, Mindrinos M, Cho RJ,
Oefner PJ, Davis RW, Ausubel FM (2000) A simple procedure for the analysis of
single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.
Plant Physiol 124:1483-1492
Moreno-Vazquez S, Ochoa OE, Faber N, Chao SM, Jacobs JME, Malsonneuve B, Kesseli
RV, Michelmore RW (2003) SNP-based codominant markers for a recessive gene
conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca
sativa). Genome 46:1059-1069
Papp AC, Pinsonneault JK, Cooke G, Sadee W (2003) Single nucleotide polymorphism
genotyping using allele-specific PCR and fluorescence melting curves.
BioTechniques 34:1068-1072
Waterfall CM, Cobb BD (2002) SNP genotyping using single-tube fluorescent
bidirectional PCR. BioTechniques 33:80-90
Programs
These are a list of the programs that I used to do sequence analysis, SNP primer design, and primer analysis
Sequence analysis:
Staden package (can be freely downloaded here)
Bioedit (can be freely downloaded here)
SNP primer design
SNP primers were designed following Drenkard et al. (above) with one exception. The authors have some very good resources on their website (here), including the web-based software for primer design called WebSNAPPER. The exception was that the reverse primer (the primer not located on the SNP) was not used. A previously designed primer was used.
All primers from WebSNAPPER were checked for primer dimers and hairpin loops both with itself and all other primers designed at each locus. The software used for this purpose was Oligo Analyzer, and the install set is difficult to find, so it is included with this webpage. It can be downloaded by clicking here. Any primer with primer dimers and/or harpin loops with a delta G less than -6 (in Oligo Analyzer) was rejected and preferences were given to primers with a delta G of secondary structures lower than -5. Delta G values of this program were consistently higher than IDT's Oligo Analyzer (listed below), but this program gives secondary structure information on multiplexed primers.
In some cases a primer had to be manually designed or modified. This was done using Primer3, a web-based primer design tool that can be found here.
Another tool that can be used to analyze primers is IDT's Oligo Analyzer (web-based) found here.
Primer testing
Primers were tested to emperically optimize the detection of SNPs. Testing methods are listed in the Protocols page.
A complete database (Miscrosoft Access file) of SNPs, their location, primers designed, primers ordered, testing of primers, etc is also included in this webpage. It may be difficult to understand Matt's methods, and no explanations are given for elements of the database, but it can be downloaded by clicking here.
SNP stability
SNP's were designed based on the fact that some mismatches are more stable than others. Here are the relative mismatches, noting that in my terminology a strong mismatch is one that will not readily pair (the two mismatched bases repel each other, making it more difficult for extension by a polymerase).
A:G strong 100
C:C strong 100
A:A strong 20 (weaker than two above)
G:G medium
C:A weak
G:T very weak
C:T very weak
T:T very weak
I pulled this information from:
Kwok S, Kellogg DE, Mckinney N, Spasic D, Goda L, Levenson C, Sninsky JJ
(1990) Effects of Primer Template Mismatches on the Polymerase Chain-Reaction -
Human-Immunodeficiency-Virus Type-1 Model Studies. Nucleic Acids Res 18:999-1005
Moreno-Vazquez S, Ochoa OE, Faber N, Chao SM, Jacobs JME, Malsonneuve B, Kesseli
RV, Michelmore RW (2003) SNP-based codominant markers for a recessive gene
conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca
sativa). Genome 46:1059-1069
This page last updated: Friday March 31, 2006 16:25