Hemi-nested touchdown PCR combined with primer-template mismatch PCR for rapid isolation and sequencing of low molecular weight glutenin subunit gene family from a hexaploid wheat BAC library
Why this work is in the frame
A frame that forgets how it found something cannot be audited. These are the routes that admitted this work.
Bibliographic record
Abstract
BACKGROUND: Hexaploid wheat (Triticum aestivum L.) possesses a large genome that contains 1.6 x 1010 bp of DNA. Isolation of a large number of gene sequences from complex gene families with a high level of gene sequence identity from genomic DNA is therefore difficult and time-consuming. Bacterial artificial chromosome (BAC) libraries can be useful for such work. Here we report on an efficient approach for rapid isolation and sequencing of the low molecular weight glutenin subunit gene family from the 'Glenlea' wheat BAC library via primer-template mismatch PCR using universal primers, primer walking using hemi-nested touchdown (TD) PCR, and followed by direct sequencing of PCR products. RESULTS: For the primer-template mismatch PCR, the universal primers were designed based on conserved gene coding regions of consensus sequences. The effects of the universal primer-template mismatches on the efficiency of standard PCR amplification were investigated after assembly of sequences from different primers amplifying the same BAC clones. Single or multiple mismatches were observed at 5' terminal, internal and the penultimate position, respectively. These mismatches included the transition mispairs G:T, T:G, A:C and the transversion mispairs A:A, A:G, G:G, G:A. Two or more primer-template mismatches reduced PCR product yield approximately from 2-fold to 10-fold compared to PCR product yield without the primer-template mismatch. For the hemi-nested TD PCR, primers were designed based on the known sequences obtained and/or published. The hemi-nested TD PCR increased both specificity and yield by high and low annealing temperatures in two consecutive amplifications. Comparison of two methods for purifying PCR products prior to sequencing showed that purification using MultiScreen384-PCR filter plates had an advantage over ethanol purification because greater numbers of sequencing reactions could be performed from comparable volumes of PCR reactions. CONCLUSION: This approach was fast, easy and cost-effective for isolation and sequencing of genes from complex gene families. It may be suitable for (i) isolation of other complex gene families and/or gene homologues from BAC libraries, (ii) for characterization of multi-copy repetitive elements pending availability of BAC libraries, and (iii) for filling in gaps in shotgun BAC sequencing.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 it