PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

Hommelsheim, Carl Maximilian; Frantzeskakis, Lamprinos; Huang, Mengmeng; Ülker, Bekir

doi:10.1038/srep05052

Cited by 105 publications

(112 citation statements)

References 41 publications

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“…The formation of artefacts, or megaprimers, that lead to the laddering effect and/or the mismatch of the desired fragment, can be caused by misalignment or slippage of the polymerase when it faces secondary structures in the sequence. Several studies suggest the hypotheses and mechanisms underlying such errors (Meyerhans et al, 1990;Sahdev et al, 2007;Hommelsheim et al, 2014;Tang and Chilkoti, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we started with the recommended method by Hommelsheim et al (2014). The authors suggested that primers that anneal far upstream or downstream of the desired sequence reduce the amount of incomplete PCR fragments.…”

Section: Discussionmentioning

confidence: 99%

“…Two more primer pairs (Table 1) were used to test the efficacy of the new program, and to test the effects of varying annealing sites on the amplification of the repetitive fragment, as reported by Hommelsheim et al (2014). The result showed that the expected fragment was amplified by both primer pairs at all DNA dilutions (Figure 8).…”

Section: Using Primers With Different Annealing Sitesmentioning

confidence: 99%

“…Recently, it was reported that amplification of repetitive sequence from transcriptional activatorlike effectors proteins is difficult and displays various sized artefacts upon electrophoresis (Hommelsheim et al, 2014), despite efforts for improvement by adding different additives to the polymerase chain reaction (PCR) mix (buffers, DMSO, and MgCl 2 ), using DNA-bindingproteins and different types of DNA polymerases, and using different annealing site primers, the most promising alternative. That report proposes that the polymerase dissociates from the template when it faces difficult structures, such as hairpin loops, in a process called slippage.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Riet¹,

Ramos²,

Lewis³

et al. 2017

Genet. Mol. Res.

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ABSTRACT. Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Using Primers With Different Annealing Sitesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Riet¹,

Ramos²,

Lewis³

et al. 2017

Genet. Mol. Res.

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show abstract

“…If a permissive promoter position has been identified a set of VarSeTALEs could be transformed into the organism of interest, rapidly producing a set of transgenic lines differing in expression of the native gene of interest. This approach would be applicable for activator or repressor dTALEs, both of which have already been used in a range of host organisms (12,35,37).…”

Section: Comparing Varsetale Design Approaches: Intra-versus Inter-rementioning

confidence: 99%

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions

et al. 2017

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Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate userdefined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33-35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base specificity and is referred to as the base specificying residue (BSR). Other repeat residues are termed non-BSRs and can contribute to TALE DNA affinity in a non-base-specific manner. Previous dTALE engineering efforts have focused on BSRs. Non-BSRs have received less attention, perhaps because there is almost no non-BSR sequence diversity in natural TALEs. However, more sequence diverse, TALE-like proteins are found in diverse bacterial clades. Here, we show that natural non-BSR sequence diversity of TALEs and TALE-likes can be used to modify DNA-binding strength in a new form of dTALE repeat array that we term variable sequence TALEs (VarSeTALEs). We generated VarSeTALE repeat modules through random assembly of repeat sequences from different origins, while holding BSR composition, and thus base preference, constant. We used two different VarSeTALE design approaches combing either whole repeats from different TALE-like sources (inter-repeat VarSeTALEs) or repeat subunits corresponding to secondary structural elements (intra-repeat VarSeTALEs). VarSeTALE proteins were assayed in bacteria, plant protoplasts and leaf tissues. In each case, VarSeTALEs activated or repressed promoters with a range of activities. Our results indicate that natural non-BSR diversity can be used to diversify the binding strengths of dTALE repeat arrays while keeping target sequences constant.

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Multivalent forms of the ribonuclease H1 hybrid binding domain are high‐affinity binders of RNA–DNA hybrids

Stopar

Nicholson

2022

FEBS Letters

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The hybrid binding domain (HBD) is a conserved fold present in ribonucleases H1 that selectively recognizes RNA-DNA hybrids, which are structures present in cellular R-loops and participate in diverse biological processes. We engineered multivalent HBD proteins to create high-affinity hybrid binders. Using EMSA-and SPR-based analyses, we showed that the triple-HBD protein exhibits a ~22 000-fold increase in hybrid affinity (K D 370 pM) relative to the single HBD (K D 8.29 lM), with the length and sequence of the linkers enabling optimal function. These findings provide a framework for testing models that correlate multivalency and affinity to understand how multivalent proteins function and also can serve to guide applications that exploit multivalency as a strategy to enhance binding affinity.

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PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

Cited by 105 publications

References 41 publications

Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions

Multivalent forms of the ribonuclease H1 hybrid binding domain are high‐affinity binders of RNA–DNA hybrids

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