A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Plan zemelʻnogo otvoda uchastka Nº 31 Krivorogskogo okruga Kazankovskogo raĭona ploshchadiʻ︠u︡ 677 gektarov

Digital image

Medida Provisória Nº 677, de 2015

Trata do aditamento de contratos de energia celebrados entre concessionárias geradoras de energia elétrica e consumidores finais e da criação do Fundo de Energia do Nordeste - FEN, autorizando a Companhia Hidro Elétrica do São Francisco - CHESF a participar do fundo.

Santos lucrou R$ 677 com bilheteria do jogo com o Grêmio, domingo

Segundo estudo da Diretoria de Análise de Políticas Públicas da FGV, o Santos lucrou apenas R$ 318 mil com seus jogos nas dez primeiras rodadas deste Campeonato Brasileiro.

[Der judišer ḥurban fun Poilen Galitzie un Bukowina fun ṭag-buch 674 - 677 (1914 - 1917)] : [1., 2. ṭeil]

Digitalisat der Ausg. Wilno {[u.a.], 1923

[Der judišer ḥurban fun Poilen Galitzie un Bukowina fun ṭag-buch 674 - 677 (1914 - 1917)] : [4. ṭeil]

Digitalisat der Ausg. Wilno {[u.a.], 1922

[Der judišer ḥurban fun Poilen Galitzie un Bukowina fun ṭag-buch 674 - 677 (1914 - 1917)] : [3. ṭeil]

Digitalisat der Ausg. Wilno {[u.a.], 1924

Na 50 Nachlass Arthur Schopenhauer - 'Schopenhauer-Archiv', 677 - Mitteilung über den Tod von Sofie Daguett und über die Erbschaftsangelegenheiten

abgedruckt in: Schopenhauer-Jahrbuch 56 (1975), S 166

Na 1 Nachlass Max Horkheimer, 677 - Forschungsprojekte über Antisemitismus, Vorurteil, Autorität: Einzeluntersuchungen (p. IX 144-IX 145)

Excerpts of Radio Talks by G. Allison Phelps, Radio Station KMTR; Institute of Social Research: An American Disciple of Adolf Hitler. A Study of the Nazi Propaganda Methods Used by Joseph E. McWilliams of the American Destiny Party in 1940 Election Campaign;

Radiolarian biostratigraphy of ODP Site 111-677

Well-preserved late Miocene through Pleistocene age radiolarian assemblages were recovered during ODP Leg 111 at Site 677, on the southern flank of the Costa Rica Rift in the eastern equatorial Pacific. Radiolarian "event" biostratigraphy (first and last morphotypic appearances) was established for Holes 677A and 677B using 21 species yielding 24 reliable datum levels. The cold upwelling waters above this site have prevented many typical tropical Pacific stratigraphic radiolarians from being useful age indicators. Biostratigraphic datum levels were assigned absolute ages based on previous work and were used to date the cores. Sedimentation rates varied from 3.7 cm/1000 yr in the late Pleistocene to 6.0 cm/1000 yr in the late Miocene. The age of the oldest sediments at this site is estimated as 5.89-6.37 Ma, which indicates that Site 677 is between magnetic anomalies 3A and 4. A total of 67 taxa were assessed for stratigraphic relevance at this site and are listed in the Appendix. One previously unknown Pliocene radiolarian stratigraphic indicator, Botryostrobus euporus (Ehrenberg), is identified.

Stable carbon and oxygen isotope record of planktonic and benthic foraminifera at ODP Site 111-677 in the Panama Basin

Oxygen and carbon isotope ratio measurements are presented for Globigerinoides ruber and for benthic species (mainly Uvigerina spp.) in the Pleistocene and uppermost Pliocene section of ODP Hole 677A in the Panama Basin. This provides the best available continuous Pleistocene stable isotope records from any location, fully justifying the recoring of DSDP Site 504. Oxygen isotope stage 22 (age about 0.85 Ma) was of similar magnitude to the most extensive glacials of the Brunhes and constitutes a logical base for the middle Pleistocene. Oxygen isotope stages as defined by Ruddiman et al. (1986, doi:10.1016/0012-821X(86)90024-5) and by Raymo et al. (1989, doi:10.1029/PA004i004p00413) back to stage 104 are recognized. Although the internationally agreed base of the Quaternary at or near stage 62 (about 1.6 Ma) is not marked by a major isotopic event, it does approximate the base of a regime characterized by highly regular 41,000-yr climate cycles. The records at Site 677 are ideal for time-series analyses and will permit a new attempt to develop a chronology for the early Pleistocene based on tuning to the orbital frequencies. The carbon isotope records also appear to contain considerable variance at orbital frequencies throughout the sequence analyzed.