PCR
Springer New York
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Timeline
Mendeley readers
| Chapter title |
Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR
|
|---|---|
| Chapter number | 4 |
| Book title |
PCR
|
| Published in |
Methods in molecular biology, May 2017
|
| DOI | 10.1007/978-1-4939-7060-5_4 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7059-9, 978-1-4939-7060-5
|
| Authors |
Maria Jorge Campos, Alberto Quesada, Campos, Maria Jorge, Quesada, Alberto |
| Editors |
Lucília Domingues |
| Abstract |
PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3'-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5'-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5'-end degenerate primers. |
Mendeley readers
The data shown below were compiled from readership statistics for 25 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 25 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 5 | 20% |
| Student > Ph. D. Student | 3 | 12% |
| Student > Bachelor | 3 | 12% |
| Student > Master | 1 | 4% |
| Professor > Associate Professor | 1 | 4% |
| Other | 0 | 0% |
| Unknown | 12 | 48% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 5 | 20% |
| Agricultural and Biological Sciences | 3 | 12% |
| Veterinary Science and Veterinary Medicine | 1 | 4% |
| Business, Management and Accounting | 1 | 4% |
| Immunology and Microbiology | 1 | 4% |
| Other | 3 | 12% |
| Unknown | 11 | 44% |