An Improved Heteroduplex Analysis for Rapid Genotyping of SNPs and Single Base Pair Indels

Fan, Jiangbo; Xia, Ye; Wang, Guo‐Liang

doi:10.2144/btn-2019-0012

Cited by 7 publications

(7 citation statements)

References 29 publications

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“…On the other hand, the larger size of the bands corresponding to heteroduplexes indicates that the loop structure affects the heteroduplex DNA mobility during electrophoresis. Other examples of decreased heteroduplexes mobility in polyacrylamide gels were shown by Fan et al (2019).…”

Section: Discussionmentioning

confidence: 85%

Four large indels detected by cpTILLING in barley chloroplast mutator seedlings

Lencina

Landau

Pacheco

et al. 2021

Preprint

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In a previous work, a polymorphism detection strategy based on mismatch digestion was applied to the chloroplast genome of barley seedlings that carried the chloroplast mutator (cpm) genotype through many generations. Sixty-two different one- or two-nucleotide- polymorphisms were detected along with four large indels: an insertion of 15 bp in the intergenic region between tRNAHis and rps19 genes, a deletion of 620 bp in the psbA gene, a deletion of 79 bp in the intergenic region between rpl33 and rps18 genes and a deletion of 45 bp in the rps3 gene. In the present investigation, we analyzed direct repeats located at the borders of those four large indels. Furthermore, we investigated the consequences of protein expression of large indels located in coding regions. The deletion of 620 bp in the psbA gene was lethal at the second leaf stage when homoplastomic. The deletion of 45 bp in the rps3 gene, which eliminates 15 amino acids, did not affect the viability of the seedlings in homoplastomy. Interestingly, the deleted segment is also lacking in the wild type version of the rps3 gene of maize and sorghum. The presence of direct repeats at the borders of the four large indels suggests that they could have originated by illegitimate recombination. This would be in agreement with a previous hypothesis that the Cpm gene product would correspond to a mismatch repair (MMR) protein devoted to maintain plastome stability by playing fundamental roles in mismatch repair during replication and avoiding illegitimate recombination.

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Section: Discussionmentioning

confidence: 85%

Four large indels detected by cpTILLING in barley chloroplast mutator seedlings

Lencina

Landau

Pacheco

et al. 2021

Preprint

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“…Although prePRIMA and iHDA are 1-bp resolution methods, a major drawback is an effort or cost to produce a probe with a few-bp deletion or insertion in the middle of a 200-bp target fragment. Unless such variants are already available, time and/or expense is required to make the probe by a two-step PCR or by cloning 29 , 30 . Alternatively, it is possible to purchase synthesized DNA, although the fee for a 200-nucleotide (nt) oligo is relatively expensive.…”

Section: Resultsmentioning

confidence: 99%

“…Such complex patterns were attributed to different conformations or different annealing possibilities of the single-stranded DNA, and may depend on experimental settings 27 . Importantly, except for a few cases 28 , it is normally impossible to distinguish 1-bp differences using HMA unless producing a double-stranded DNA probe with mutations (called improved heteroduplex analysis (iHDA) 29 ). Here, we developed a generally applicable method to detect a 1-bp difference using a short single-strand DNA (sssDNA) probe and called it PRIMA ( Pr obe- I nduced H MA , Fig.…”

Section: Introductionmentioning

confidence: 99%

PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes

Kakui

Yamazaki

Shimizu

2021

Sci Rep

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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.

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“…Heteroduplex Mobility Assay (HMA) can also distinguish between the heteroduplexes and homoduplexes DNA in native PAGE but indels smaller than 3 bp in size can be missed 11 , 12 . Improved versions of the HMA called improved heteroduplex analysis (iHDA) 13 and Probe-Induced HMA (PRIMA) 14 use DNA probes to detect 1-bp pair indels after gene editing. Though these techniques can detect a wide range of indels, they does involve multiple steps.…”

Section: Introductionmentioning

confidence: 99%

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

Ijaz

Nakazato

Setou

et al. 2022

Sci Rep

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The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using “face-to-face” primers where the 3’ ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.

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An Improved Heteroduplex Analysis for Rapid Genotyping of SNPs and Single Base Pair Indels

Cited by 7 publications

References 29 publications

Four large indels detected by cpTILLING in barley chloroplast mutator seedlings

Four large indels detected by cpTILLING in barley chloroplast mutator seedlings

PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

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