SOP³: A Web-Based Tool for Selection of Oligonucleotide Primers for Single Nucleotide Polymorphism Analysis by Pyrosequencing®

Figures & data

Figure 1. Representation of the organization of the SOP³application web site.

Data were combined from the University of California Santa Cruz (UCSC) Genome Browser (Build 34) and dbSNP (Build 118) from the National Center for Biotechnology Information (NCBI) into the SOP³ database. The SOP³ application enables the analysis of SNP attributes, including flanking sequence and chromosomal location, during the design of primer sets for PCR and Pyrosequencing. SNP, single nucleotide polymorphism.

Table 1. Inputs, Default Values, and Output for SOP³

Input	Default Value
Search Settings
Entry box for gene name (locus) or RefSeq	gene name (locus)
Number or chromosomal location
PCR Primer Design
Tm^a	60°C
Flank length (maximum 1000 each side)	1000nucleotides
Use repeat masker (yes or no)	no
Use extra 5′ nucleotide	yes
Maximum PCR size	500 nucleotides
Minimum foldover	5 nucleotides
Unique n-mer length	6 nucleotides
AT test (yes or no)	no
Residue occurrence thresholds (minimum and maximum)	minimum 14, maximum 40
Pyrosequencing Primer Design
Tm^a	40°C
Primer minimum distance from SNP	1 nucleotide
Maximum distance from SNP	30 nucleotides
Function Type
Can limit search by specific SNP attributes such as:	all selected
UTR, splice site, synonymous or nonsynonymous
change, coding region, or intron.
Order Results and Save
Results can be saved to file (yes or no)	no
Results can be parsed by 1 or 2 limits:	heterozygosity, ascending
(heterozyosity, gene name, rs#, location,
chromosome, or function).
Ouput Display
Gene name, rs#, and residue change
Heterozygosity value, primer sequences
Nucleotide sequence flanking SNP
Sites of PCR primer annealing	Highlighted in yellow
Sites of Pyrosequencing primer annealing	Highlighted in red
Location of nearby SNPs	Highlighted in blue
Repeat masked regions	Capitalized, highlighted in gray
Predicted program (yes or no)	No

T_m, melting temperature; SNP, single nucleotide polymorphism; UTR, untranslated region; rs#, reference sequence number.

^a T_m was calculated using the equation T_m=16.6*log[cation concentration]+41 ^*(fraction of GC)+81.5, where the concentration of cation was estimated to be 0.1 M (14).

Figure 2. Web interface of the output page of the SOP³primer design application.

The primer set was designed for SNP rs3842748 from the human insulin gene. The gene name, SNP reference sequence number, allele description, average heterozygosity value, and location of the SNP are indicated in the upper part of the figure. Primers for Pyrosequencing and PCR, along with the calculated length of the PCR amplification product, are given in the central part of the figure. A nucleotide motif at the 5′ end of the forward PCR primer, separated by a space from the rest of the primer sequence, signifies the modification to the primer sequence to avoid self-priming during Pyrosequencing. In the lower area of the figure, the chromosomal DNA sequence surrounding the SNP is illustrated. The polymorphic residues are shaded blue while primers for PCR and Pyrosequencing are shaded yellow and red, respectively. SNP, single nucleotide polymorphism.

Figure 3. Calculated Pyrosequencing results for human insulin-associated SNP rs3842748.

Oligonucleotide primers for PCR and Pyrosequencing are indicated in Figure 2. The SOP³ application provided a recommended order of nucleotide dispensation events and pyrogram for each (A and B) homozygous and (C) heterozygous genotype. The nucleotide sequence examined is indicated in each panel. SNP, single nucleotide polymorphism.

Figure 4. Observed Pyrosequencing results for SNP rs2056402.

Primers for PCR and Pyrosequencing were chosen using the SOP³ application. Genomic DNA was obtained from healthy donor volunteers. Samples genotyped were (A) homozygous AA, (B) homozygous TT, and (C) heterozygous. The nucleotide dispensation order is indicated along the x-axis while the Pyrosequence is illustrated for each sample. Nucleotide disposition A6, following the polymorphic residues, was intended to serve as a negative control for background signal. SNP, single nucleotide polymorphism.

Table 2. Selected SNPs Developed for Pyrosequence-Based Typing Assays Using the SOP³ Primer Design Application

RefSeq	Gene Name	Description	Chromosome	Gene Element ^a	Alleles	Heterozygosity
rs913746	COL4A2	Collagen, typeIV, a2	13	Intron	AC	0.463
rs2272946	COL4A5	Collagen, typeIV, a5	X	Exon (nonsyn)	GT	Not reported
rs3747408	COL4A5	Collagen, typeIV, a5	X	Exon (syn)	AG	Not reported
rs2056402	ITGA2	Integrin,a2	5	Intron	AT	0.198
rs3212504	ITGA2	Integrin, a2	5	Intron	CG	0.326
rs27890	ITGA2	Integrin, a2	5	Intron	CT	0.203
rs26679	ITGA2	Integrin, a2	5	Intron	CG	0.5
rs1421941	ITGA2	Integrin, a2	5	Intron	CT	0.395
rs3212594	ITGA2	Integrin, a2	5	Exon (syn)	AG	0.192
rs17468	ITGB1	Integrin, 1	10	UTR	CT	0.413
rs1316757	ITGB1	Integrin, 1	10	Intron	AC	0.418
rs2230396	ITGB1	Integrin, 1	10	Exon (syn)	AC	0.187

SNP, single nucleotide polymorphism.

^a Gene element symbols are synonymous (syn), nonsynonymous (nonsyn), and untranslated region (UTR).