ArticleNucleic acids research2003
Regionalized GC content of template DNA as a predictor of PCR success.
Article in Nucleic acids research, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 129 citations in OpenAlex.
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- Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses.Microbiome · 2018Article
- Illumina Library Preparation for Sequencing the GC-Rich Fraction of Heterogeneous Genomic DNA.Genome biology and evolution · 2018Article
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Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A set of 1438 human exons was subjected to nested PCR. The initial success rate using a standard PCR protocol required for ligation-independent cloning was 83.4%. Logistic regression analysis was conducted on 27 primer- and template-related characteristics, of which most could be ignored apart from those related to the GC content of the template. Overall GC content of the template was a good predictor for PCR success; however, specificity and sensitivity values for predicted outcome were improved to 84.3 and 94.8%, respectively, when regionalized GC content was employed. This represented a significant improvement in predictability with respect to GC content alone (P < 0.001; chi(2)) and is expected to increase in relative sensitivity as template size increases. Regionalized GC was calculated with respect to a threshold of 61% GC content and a sliding window of 21 bp across the target sequence. Fine-tuning of PCR conditions is not practicable for all target sequences whenever a large number of genes of different lengths and GC content are to be amplified in parallel, particularly if total open reading frame or domain coverage is essential for recombinant protein synthesis. Thus, the present method is proposed as a means of grouping subsets of genes possessing potentially difficult target sequences so that PCR conditions can be optimized separately in order to obtain improved outcomes.
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Registered trials
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