Secondary structure within PCR target sequences may facilitate heteroduplex production.

Separovic, Evica Rajcan; Nadin-Davis, Susan A.

doi:10.1101/gr.3.4.248

Cited by 7 publications

(3 citation statements)

References 8 publications

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“…2, 3, and 9) showed distinct doublet bands. PCR amplification using a mixture of templates with and without an insertion has been previously shown to result in the production of heteroduplex PCR products showing anomalous electrophoretic migration (Separovic and Nadin-Davis, 1994). We observed that, as expected, PCR products spanning exons 2-6 generated from purified IIA or IID templates migrated similarly on 2% agarose gels (Fig.…”

Section: Discussionsupporting

confidence: 68%

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation

et al. 2008

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Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow-derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by the use of an alternative 5' splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by the use of another 5' alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5' splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites.

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Section: Discussionsupporting

confidence: 68%

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation

et al. 2008

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“…The frequency of heteroduplex formation (4,8,19) would be expected to increase in the later cycles of mixed PCR amplifications, when the amplified DNA species reach concentrations high enough to compete with primers for binding sites (23). Cloned heteroduplex molecules may be subjected to E. coli DNA repair mechanisms (2,3,11,17,18), resulting in hybrid plasmid inserts.…”

mentioning

confidence: 99%

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

Speksnijder

Kowalchuk

Jong

et al. 2001

Appl Environ Microbiol

210

139

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A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

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“…In our study, unexpected patterns were detected only when two different SSU rDNA sequences were mixed, amplified and cloned (Table 3), and the rate of artifact clones was 22%. In addition, artifact clones increased when PCR was done with a number of cycles higher than 25 as described previously in studies on factors influencing the generation of heteroduplexes or chimeric molecules [28,48,581. Such hybrid DNA formation may occur when amplifying target sequences that are members of a gene family [49] or derived from related organisms [21, 521.…”

Section: Discussionmentioning

confidence: 99%

Diversity In Symbiotic Dinoflagellates (Pyrrhophyta) from Seven Scleractinian Coral Species: Restriction Enzyme Analysis of Small Subunit Ribosomal RNA Genes

Darius

Dauga

Grimont

et al. 1998

J Eukaryotic Microbiology

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The diversity of symbiotic dinoflagellates (SD) from seven coral species (Fungia scutaria, Fungia paumotensis, Lep‐tastrea transversa, Pavona cactus, Pocillopora verrucosa, Montastrea curia, and Acropora fonnosa) was studied in a restricted geographical area, the Lagoon of Arue on the island of Tahiti. Their diversity was explored by small subunit ribosomal RNA gene (SSU rDNA) restriction fragment length polymorphism (RFLP). After a nested amplification with SD specific primers, RFLP analyses were performed directly and after a cloning step. The diversity of these different SSU rDNA was estimated in respect to possible technical artifacts. In an axenic culture of SD from the coral Galaxea fascicularis, both heterogeneous SSU rDNAs and artifact molecules were observed as in our SD samples. According to the number of patterns observed, corals Fungia paumotensis, Leptastrea transversa. Pavona cactus, Montastrea curia, and Acropora fonnosa contained one class of SD SSU rDNAs. whereas Fungia scutaria and Pocillopora verrucosa contained three and two classes of SD SSU rDNAs respectively. In the limited geographic area studied. SD from different coral species shared the same pattern, except SD from Montastrea curta, which showed a unique pattern. In addition to the possibility of SD flux among different coral species, specific mechanisms could also be involved in the establishment of a symbiosis.

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Secondary structure within PCR target sequences may facilitate heteroduplex production.

Cited by 7 publications

References 8 publications

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

Diversity In Symbiotic Dinoflagellates (Pyrrhophyta) from Seven Scleractinian Coral Species: Restriction Enzyme Analysis of Small Subunit Ribosomal RNA Genes

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