Single nucleotide insertions or deletions drastically disrupt primer annealing
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The retrieved literature discusses small nucleotide insertions, deletions, and replication errors, but does not fully substantiate the broad claim that single nucleotide insertions or deletions drastically disrupt primer annealing in all contexts.
With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans. Each SS primer consists of a 5′ “anchor” that hybridizes to the template, followed by a noncomplementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.
Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1-5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3' end are partially corrected and extended. Indels located 7-9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.
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