Abstract
Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. Although several methods have been developed to detect such 1-bp indels, each method has pros and cons in terms of cost and/or resolution. Heteroduplex mobility assay (HMA) is a traditional technique detecting small base pair differences but it has a limited resolution of mutation size and the band patterns are often complex. Here, we developed a new method called PRIMA (Probe-Induced HMA) using a short single-stranded DNA molecule as a probe in HMA. By utilizing a 40-mer probe containing a 5-nucleotide deletion, we assessed the mobility of a heteroduplex with a target DNA fragment from a plant, bacterium, and human. This method allowed us to detect a 1-bp indel mutation consistently. We also showed that SNPs can be detected using PRIMA. PRIMA provides a rapid and cost-effective solution for the genotyping.
Highlights
Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair insertion or deletion mutations
Methods detecting small base pair differences have been developed by many researchers[15], for example, Sanger or deep sequencing, restriction fragment length polymorphism (RFLP) a nalysis[16], heteroduplex mobility assay (HMA)[17,18,19,20], DNA melting a nalysis[21], T7 endonuclease I assay[22], Cel-1 assay[23], fluorescent polymerase chain reaction (PCR)[24], and RNA-guided engineered nuclease (RGEN)-RFLP25
Heteroduplex mobility assay (HMA) consists of four simple steps: (1) PCR
Summary
Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. There is an increasing demand in molecular biology to detect 1-bp differences because of the recent advancement of gene-editing technology (ZFN/TALEN/CRISPR) based on double-strand breaks (DSBs) These DSBs can stimulate non-homologous end joining (NHEJ) at the targeted genome sequence and often produce a 1-bp insertion or deletion (indel) mutation[1,2,3,4,5]. Methods detecting small base pair differences have been developed by many researchers[15], for example, Sanger or deep sequencing, restriction fragment length polymorphism (RFLP) a nalysis[16], heteroduplex mobility assay (HMA)[17,18,19,20], DNA melting a nalysis[21], T7 endonuclease I assay[22], Cel-1 assay[23], fluorescent polymerase chain reaction (PCR)[24], and RNA-guided engineered nuclease (RGEN)-RFLP25. We describe PRIMA, which has 1-bp resolution and is a fast method capable of genotyping
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