Single nucleotide polymorphism genotyping using locked nucleic acid (LNA™)

Mouritzen, Peter; Nielsen, Alex Toftgaard; Pfundheller, Henrik M.; Choleva, Yoanna; Kongsbak, Lars; Möller, Sören

doi:10.1586/14737159.3.1.27

Cited by 106 publications

(102 citation statements)

References 21 publications

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“…72 Furthermore, LNAs display excellent mismatch discrimination. Mouritzen et al 73 showed single-nucleotide specificity against complementary DNA using fully modified 12 nt LNA probes coupled to glass slides during the development of a microarray used to probe samples for single-nucleotide polymorphisms (SNPs) associated with human dysmetabolic syndrome. LNA oligonucleotides have not been tested in humans, but results in rodents are promising.…”

Section: Locked Nucleic Acid Amosmentioning

confidence: 99%

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

2005

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Section: Locked Nucleic Acid Amosmentioning

confidence: 99%

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

2005

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“…Eukaryotic b-actin mRNA was used as reference gene to normalize for differences in the amount of total RNA in each sample. All probes were purchased from Exiqon (Vedbaek, Denmark) (Mouritzen et al, 2003) (Supplementary Table 1). All calculations were based on the mean value of PCR reactions performed in triplicate.…”

Section: Real-time Quantitative Pcrmentioning

confidence: 99%

Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-κB and ATF4

et al. 2008

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Under anoxia a coordinated, cytoprotective program is induced, called the unfolded protein response (UPR). Activating transcription factor 4 (ATF4) is a mediator of the UPR and activates a gene expression program, promoting tumour growth and survival under anoxia. A key gene induced by ATF4 under normoxic conditions is SKIP3. We characterized the induction of SKIP3 during anoxic exposure to determine whether UPR alone was sufficient or there was a more complex regulatory response to anoxia. There was temporal separation of acute hypoxia-inducible factor (HIF)-1a-and chronic ATF4-dependent gene expression programs. SKIP3 was regulated by chronic (48 h) rather than acute anoxia (o24 h) by a complex set of pathways and mechanisms, besides ATF4 induced by the classical UPR, there was transcriptional regulation by nuclear factor-kappa B (NFjB) and RNA stabilization by HuR. Temporal activation of the NF-jB pathway under anoxia protected cells from negative consequences of the oxygen stress and involved the canonical signalling pathways that promote IjBA phosphorylation and degradation, and reduced mRNA level of the inhibitory protein IjBA followed by the translational repression of IjBA. We also show that SKIP3 acts as an inhibitor of NF-jB and ATF4-dependent transcription under anoxia and provides a regulatory feedback loop. Repression of the survival pathway NF-jB by SKIP3 sensitized cells to metabolic consequences of the anoxic stress. Thus, the response to anoxia is mediated by three pathways independently of HIF, suggesting that combined therapeutic approaches would be needed to maximize effects against this pathway.

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“…The strong affinity of LNA substituted oligonucleotides for complementary sequences results in high melting points. However, a one base mismatch between an LNA oligonucleotide and the complementary strand can lower the melting point 20-301C, thereby allowing an LNA oligonucleotide to discriminate a one base pair difference between templates (Mouritzen, 2003). For example, a DNA octomer of bases TGCTGGTG has a Tm of 351C, whereas the corresponding LNA octomer exhibits a Tm of 711C.…”

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confidence: 99%

“…For example, a DNA octomer of bases TGCTGGTG has a Tm of 351C, whereas the corresponding LNA octomer exhibits a Tm of 711C. A single base pair mismatch in the DNA octomer decreases Tm by 101C; whereas a single base pair mismatch in the LNA octomer creates a DTm of 261C (Mouritzen, 2003).…”

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confidence: 99%

Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens

Dominguez

Kolodney

2005

Oncogene

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Detection and sequencing of mutations from clinical specimens is often complicated by the presence of an excess of nonmutated cells. To facilitate the detection and sequencing of minority mutations from clinical specimens, we developed wild-type blocking polymerase chain reaction (WTB-PCR). This technique allows sensitive detection of minority mutations in a tissue sample containing excess wild-type DNA. In WTB-PCR, a nonextendable locked nucleic acid (LNA) oligonucleotide binds tightly to a region of wild-type DNA known to develop point mutations. This LNA sequence blocks amplification of wild-type DNA during PCR while permitting amplification of mutant exon 15. Our results show that the LNA blocking oligonucleotide inhibits amplification of wildtype DNA in a dose-dependent manner. WTB-PCR was able to detect mutant DNA in clinical samples of melanoma tissue containing an excess of nonmelanoma cells. This method was also able to detect small amounts of point mutated or tandem mutated DNA diluted with a much larger concentration of wild-type DNA. This rapid and simple assay overcomes the limitations of current methods to detect minority mutations. The potential applications of WTB-PCR include early diagnosis and prognosis of various cancers.

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Single nucleotide polymorphism genotyping using locked nucleic acid (LNA™)

Cited by 106 publications

References 21 publications

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-κB and ATF4

Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens

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