High-throughput Primer Design Research Articles

High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach.

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR. Unfortunately, this approach is very often coupled with PCR artifacts which compromise overall efficiency of site-directed mutagenesis. To reduce the failure rate due to the PCR artifacts, we have set up a high-throughput mutagenesis protocol based on a two-fragment PCR approach. To this end, each mutation is introduced in two separate PCRs resulting in two linear fragments of the mutated plasmid. In the next steps, the PCR template is digested and the two matching plasmid fragments are joined together using Gibson assembly. Separating the corresponding mutagenic primers into two different PCRs decreases a number of artifacts and thus increases overall cloning efficiency. Furthermore, free software that we developed facilitates both high-throughput primer design and analysis of sequencing results. Overall, this protocol enabled us to efficiently produce several alanine-scanning libraries of 400 single-point mutations with complete coverage of the protein sequence.

PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR

The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously ( PrimerSuite ), (ii) screen for primer-primer dimerizing artefacts ( PrimerDimer ), and (iii) support multiplex PCR assays ( PrimerPlex ). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).

PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection.

Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction.

PrimerView: high-throughput primer design and visualization.

BackgroundHigh-throughput primer design is routinely performed in a wide number of molecular applications including genotyping specimens using traditional PCR techniques as well as assembly PCR, nested PCR, and primer walking experiments. Batch primer design is also required in validation experiments from RNA-seq transcriptome sequencing projects, as well as in generating probes for microarray experiments. The growing popularity of next generation sequencing and microarray technology has created a greater need for more primer design tools to validate large numbers of candidate genes and markers.ResultsTo meet these demands I here present a tool called PrimerView that designs forward and reverse primers from multi-sequence datasets, and generates graphical outputs that map the position and distribution of primers to the target sequence. This module operates from the command-line and can collect user-defined input for the design phase of each primer.ConclusionsPrimerView is a straightforward to use module that implements a primer design algorithm to return forward and reverse primers from any number of FASTA formatted sequences to generate text based output of the features for each primer, and also graphical outputs that map the designed primers to the target sequence. PrimerView is freely available without restrictions.

Development of a next‐generation sequencing platform for retinal dystrophies, with LCA and RP as proof of concept

Abstract Purpose Retinal dystrophies represent an emerging group of hereditary disorders that lead to degeneration of the photoreceptors and/or the retinal pigment epithelium, resulting in irreversible blindness. They are genetically complex, with over 200 disease loci identified so far. Current genetic screening consists of microarray analysis (Asper Ophthalmics) for the most recurrent mutations, and subsequent Sanger sequencing. However, the high cost and low throughput of the latter technology limits testing to only the most recurrent genes. This project aims to develop a high throughput and cost‐effective platform for screening of all known disease genes for Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP), using the next‐generation sequencing (NGS) technology. Methods A NGS panel will be developed for all 16 and 47 known LCA and RP genes, respectively, including coding and untranslated regions, regulatory regions and microRNA binding sites. The protocol will consist of the following steps: 1) high throughput primerdesign and qPCR, 2) ligation, 3) shearing and 4) sequencing on the Illumina Genome Analyser IIx (GAIIx). This innovative protocol overcomes the need for short amplicons in order to render short‐read sequences by the GAIIx. This sequencing instrument was chosen because of its high capacity, low cost per base and the absence of interpretation problems at homopolymeric regions. Analysis of the variants will be performed using in‐house developed and commercial software, which ranks all variants according to their pathogenic potential. Conclusion Using the proposed protocol, comprehensive screening for all known disease genes for LCA and RP will be available for the first time.