Pcr in clinical diagnosis

Ronai, Ze’ev A.; Yakubovskaya, Marina S.

doi:10.1002/jcla.1860090409

Cited by 15 publications

(7 citation statements)

References 170 publications

(49 reference statements)

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“…This may be explained by the use of optimal PCR conditions for the amplification until the last stage and by the short incubation at 72 8C at the very end when the Taq DNA polymerase elongates DNA to perfect duplex [2]. We did not notice the appearance of additional bands immediately after PCR.…”

Section: Tetramer Formation In Different Conditionsmentioning

confidence: 74%

Interaction of linear homologous DNA duplexes via Holliday junction formation

Yakubovskaya¹,

Neschastnova²,

Humphrey³

et al. 2001

European Journal of Biochemistry

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Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.

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Section: Tetramer Formation In Different Conditionsmentioning

confidence: 74%

Interaction of linear homologous DNA duplexes via Holliday junction formation

Yakubovskaya¹,

Neschastnova²,

Humphrey³

et al. 2001

European Journal of Biochemistry

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“…This has become possible through the development of highly sensitive PCR-based methodologies which selectively amplify the mutant even if its incidence is 1/10-4 to These methodologies include the mismatch amplification mutation assay (Cha et al, 1992), the enriched PCR (EPCR; Kahn et a/., 1991; and other PCR-based cloning and hybridization processes with selective probes (Ma0 and Sidransky, 1994). Comparisons of these methodologies have been reviewed (Ronai and Yakubovskaya, 1995). Early detection of mutation has been shown for the ras oncogene in the case of colon and pancreatic cancer.…”

mentioning

confidence: 99%

High frequency of K‐ras mutations in normal appearing lung tissues and sputum of patients with lung cancer

Yakubovskaya¹,

Spiegelman²,

Luo

et al. 1995

Intl Journal of Cancer

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To evaluate the possible use of mutant ras as a biomarker for lung cancer, we have analyzed "normal appearing" lung tissue, lung tumor, lung metastases and sputum samples from patients with non-small cell lung cancer (NSCLC). As a control, we used lung tissue and sputum samples from patients without oncological diseases or lung disorders. Our analyses were performed with the aid of enriched PCR (EPCR), a method which enables detection of ras mutation even if present at low incidence. EPCR identified K-ras codon 12 mutations in 10% of lung tissues obtained from patients with no lung diseases, whereas the same mutation was detected in 60% of samples of normal appearing lung tissues obtained from patients with NSCLC, 62% of NSCLC tumors and 80% of metastases. Analysis of sputum samples of patients with NSCLC identified 47% to harbor mutant ras allele, whereas 12.5% of controls diagnosed with non-oncological lung diseases carried this mutation. Most of these mutations were detected with the aid of EPCR only, indicating that a minority of cells in a given sample harbor this mutation. The ability to detect K-ras codon 12 mutation in 60% of lung tissue samples and in 47% of sputum samples taken from patients with lung cancer (as compared with 10% and 12.5% of respective controls) points to the potential use of ras mutation as a biomarker for exposure and possible identification of patients who may be at higher risk of developing lung cancer.

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“…The one was magnifying the signal by giving up it obtained conventionally from short hybrid region but using the signal from other region. The other was the adopting of PCR, by which a trace amount DNA can be amplified to mass quantity of DNA (Ronai and Yakubovskaya, 1995;Gurvitz et al, 1994;Scheu et al, 1998;Mazars et al, 1991;Isacsson et al, 2000).…”

Section: Detection Limitmentioning

confidence: 99%