SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design

Abstract

The Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a relatively simple and inexpensive method for genotyping single nucleotide polymorphisms (SNPs). It requires minimal investment in instrumentation. Here, we describe a web application, 'SNP Cutter,' which designs PCR-RFLP assays on a batch of SNPs from the human genome. NCBI dbSNP rs IDs or formatted SNPs are submitted into the SNP Cutter which then uses restriction enzymes from a pre-selected list to perform enzyme selection. The program is capable of designing primers for either natural PCR-RFLP or mismatch PCR-RFLP, depending on the SNP sequence data. SNP Cutter generates the information needed to evaluate and perform genotyping experiments, including a PCR primers list, sizes of original amplicons and different allelic fragment after enzyme digestion. Some output data is tab-delimited, therefore suitable for database archiving. The SNP Cut-ter is available at http://bioinfo.bsd.uchicago.edu/SNP_cutter.htm.

Figure 1

SNP Cutter workflow. Rectangles and rhombi show the task that the program performs. It includes five steps, shown as Step 1 through 5 in the figure. Parallelograms show the data that the user supplies or every step produces. The program produces five files as shown. ‘(tag.)sc.xxxx_DNinput.txt’ is the output of Step 1 SNPSequer; ‘(tag.)sc.xxxx_PRI3.txt’ is the output of Step 3 of Primer3 analysis; ‘(tag.)sc.xxxx_detail.txt’ and ‘(tag.)sc.xxxx_rflp.txt’ are the outputs of Step 5. ‘(tag.)sc.xxxx_failed.txt’ logs all SNPs failed at assay design. ‘RE’ is the abbreviation for ‘Restriction Enzyme.’ ‘(tag.)’ is an optional string user provides as identifier in the filename. ‘xxxx’ is a random number that the system produces.

Figure 2

Illustration of an example of detail data file. All the information about the primers, amplicons, enzymes and fragments are shown in this file. The real sequence data of ‘Amplicon1_seq’ and ‘Amplicon2_seq’ were omitted to save space.