METHYLENETETRAHYDROFOLATE REDUCTASE (MTHFR) MULTIPLEX – PCR METHOD
Bibliographic record
Abstract
Introduction Gene polymorphisms in human methylenetetrahydrofolate reductase (MTHFR) have been linked to the disorders of folate metabolism, and may contribute to such disease as neural tube defect, coronary heart disease, venous thrombosis, and several types of cancers. Methodology We have modified our current real-time PCR method (Clin Biochem 2000;33:535-539.) for the detection of the MTHFR c.677C > T variant to a real-time multiplex-PCR method for detection of MTHFR c.1298A > C and c.677C > T variants on a LightCycler 1.2. Our multiplex method is partly based on that of Agarwal et al.(J Mol Diag 2007;9:345). Results The genotyping results of c.677C > T variant analyzed by both methods were in agreement. All samples analyzed for the c.1298A > C variant by the Multiplex-PCR method gave clear-cut results. The LightCycler multiplex method was validated with 14 specimens previously genotyped by restriction fragment length polymorphism at Montreal Children’s Hospital. All results were in agreement except for one sample that failed to amplify. There was no significant difference in Tm between the Multiplex-PCR procedure and the previously reported Tm’s for both variants. When run alone but not as a multiplex, the progress curve of the c.1298A > C variant exhibited the “hook effect”. Asymmetric amplification was found to reduce this effect. Conclusions The multiplex assay is reliable, economical, fast and simple method to perform as compared with RFLP technique. Asymmetric PCR is a helpful tool to minimize the hook effect.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.003 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.003 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.005 | 0.006 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".