Abstract
Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to ‘stitch’ individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user’s input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.
Highlights
Input for STITCHER 2.0 is a single sequence in fastA format[6]
During primer design STITCHER 2.0 will move along in increments defined by the user within the specified 5′and 3′search areas
To validate primers designed using STITCHER 2.0 a series of polymerase chain reaction (PCR) reactions using primers designed by STITCHER 2.0 that span the Caenorhabditis elegans gene ama-1 were performed using the following cycling conditions: 1) 95 °C for 5 mins; 2) 95 °C for 30 secs; 3) 57 °C for 30 secs; 4) 72 °C for 1 min, with steps 2 to 4 repeated 35 times, followed by 72 °C extension for 10 mins
Summary
Input for STITCHER 2.0 is a single sequence in fastA format[6]. Once a sequence is inserted, the user completes the Primer Design Parameters section. By default, both forward and reverse primers are designed, but either can be deselected. The 5′and 3′search areas are specified by the user followed by the maximum and minimum percentage GC and primer Tm values for the forward and reverse primers. The addition of a GC clamp to either the forward or reverse primer can be specified (by default this is set to ‘No’). During primer design STITCHER 2.0 will move along in increments defined by the user within the specified 5′and 3′search areas. The default increment is 2 bp, the forward and reverse
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