An error prone PCR method for small amplicons

Lee, Sea On; Fried, Stephen D.

doi:10.1016/j.ab.2021.114266

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…We synthesized all four Alu sequences and cloned them into the Alu jumping vector followed by Sanger sequence validation. We then used an error-prone PCR system that is estimated to generate a mutation every 1-16bp per kilobase (kb) of DNA (see Methods) to generate roughly 1-6 mutations per 300bp Alu element 10,11 . The mutagenized PCR product of each Alu element was then re-cloned into the Alu jumping assay vector to generate four mutagenized libraries: Alu Sx-mut, Alu 6b-mut, Alu 14b-mut and Alu h1.1-mut ( Supplementary Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

Massively parallel jumping assay decodesAluretrotransposition activity

Matharu,

Zhao,

Sohota

et al. 2024

Preprint

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The human genome contains millions of retrotransposons, several of which could become active due to somatic mutations having phenotypic consequences, including disease. However, it is not thoroughly understood how nucleotide changes in retrotransposons affect their jumping activity. Here, we developed a novel massively parallel jumping assay (MPJA) that can test the jumping potential of thousands of transposons en masse. We generated nucleotide variant library of selected four Alu retrotransposons containing 165,087 different haplotypes and tested them for their jumping ability using MPJA. We found 66,821 unique jumping haplotypes, allowing us to pinpoint domains and variants vital for transposition. Mapping these variants to the Alu-RNA secondary structure revealed stem-loop features that contribute to jumping potential. Combined, our work provides a novel high-throughput assay that assesses the ability of retrotransposons to jump and identifies nucleotide changes that have the potential to reactivate them in the human genome.

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Section: Resultsmentioning

confidence: 99%

Massively parallel jumping assay decodesAluretrotransposition activity

Matharu,

Zhao,

Sohota

et al. 2024

Preprint

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“…We found that conventional error-prone PCR methods were ill-suited for achieving the desired mutation density in this small amplicon (1–2 mutations per 36 bp) while also maintaining low levels of bias; − hence, we developed a modified error-prone PCR method based on iterating between dilution and reamplification with error-prone touchdown PCR to suppress accumulation of incorrect products. The details of this modified method are reported elsewhere …”

Section: Resultsmentioning

confidence: 99%

“…The starting point for these mutations was the pBAD-tdTEVDB variant with the trimmed-down context regions (DVFLG deleted from the 5′-exon context and PFNI deleted from the 3′-exon context, see Figure 5 ). The commercial GeneMorph II Random Mutagenesis kit (Agilent) was used to perform epPCR, but using a modified protocol that involved performing several iterations of dilution and reamplification with a touchdown PCR protocol (see ref ( 51 ) for details). The forward and reverse primers SD_lib_insert_F/R ( Table S1 and Figure S10 ) were designed to span the initiation sequence.…”

Section: Methodsmentioning

confidence: 99%

Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins

2021

Self Cite

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The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA in vivo by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling “loopable” translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.

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“…Traditionally, this type of construct could be possibly made by regular PCRs (generating the constant fragments) and error-prone PCRs (generating the mutational fragments) followed by some kinds of gene-assembly methods, such as the Golden Gate assembly or yeast homologous recombination , (Figure S15). However, there are several obstacles that render the traditional method impractical, especially for short mutational fragments (such as CDRs ∼10–60 bp): (i) more PCRs required for the traditional method (see Figure S15); (ii) difficulty to generate small amplicons by error-prone PCRs; and (iii) low efficiency by yeast homologous recombination or the Golden Gate assembly, especially for a large number of fragments or small fragments. Therefore, the reverse Kunkel method provides a unique strength for engineering biomolecules whose functions rely on multiple discontinuous short sequences, such as the CDRs in antibodies or alternative affinity scaffolds, the hairpins in aptamers or ribozymes, the hypervariable regions in T cell receptors or AAV capsids, the RuvC domain in Cas9 or Cas12a, and so forth.…”

Section: Discussionmentioning

confidence: 99%

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method

et al. 2022

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Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

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An error prone PCR method for small amplicons

Cited by 7 publications

References 27 publications

Massively parallel jumping assay decodesAluretrotransposition activity

Massively parallel jumping assay decodesAluretrotransposition activity

Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method

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