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A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Ling, Michael Mingfu Merante, Frank Robinson, B. H. |
| Copyright Year | 1995 |
| Abstract | Yeast is an ideal host system for studying exogenous eukaryotic gene expression and for studying gene structures from recombinant plasmid DNAs and yeast artificial chromosomes (YACs). Many yeast expression studies may also require the altering of the genome itself by homologous recombination. All these studies require the screening for positive transformants and the most popularly used method of screening is PCR. As some gene replacement experiments have a low success rate (as low as 4%) (1), it becomes necessary to screen a large number of clones. Hundreds and thousands of transformants, e.g. 1200 YAC clones/ day/lab as reported by Khristich et al. (2), are screened by PCR in many laboratories. Conventionally, PCR is carried out with purified DNAs, i.e. plasmids (3), YACs (4) or genomic DNAs (5) from yeast. The purification of DNAs involves either the use of glass beads to disrupt intact cells or the use of lytic enzymes to digest cell walls, followed by numerous inconvenient steps of organic solvent extraction and alcohol precipitation. These purification methods are time-consuming (2-5 h for <24 samples) and require a significant amount of starting culture. With a generation time of 100-300 min, single colonies of yeast require an additional 1-3 days to grow to the late log phase in 1-15 ml culture to provide sufficient amounts of cells for manipulation during the purification. A previous report showed that fresh yeast colonies can be used directly, without prior DNA purification, for PCR to obtain a 1.1-kb genomic PCR product (6). However, another group reported that direct PCR with fresh yeast colonies could not even detect products amplifiable from plasmid DNAs (7). In fact, plasmid DNAs are easier to isolate than genomic DNAs and plasmid DNAs can be used in higher molar concentrations for PCR. Some researchers have success in boiling yeast colonies in a small volume of water for 5-10 min, and using either the boiled cells directly or the supernatant from a brief spinning of the boiled cells as a plasmid or YAC source for PCR (unpublished results and personal communications). Our repeated trials using these two methods could not consistently yield genomic PCR products ranging from 0.3 to 3 kb in size. |
| File Format | PDF HTM / HTML |
| DOI | 10.1093/nar/23.23.4924 |
| PubMed reference number | 8532539 |
| Journal | Medline |
| Volume Number | 23 |
| Issue Number | 23 |
| Alternate Webpage(s) | https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/nar/23/23/10.1093_nar_23.23.4924/2/23-23-4924.pdf?Expires=1492552077&Key-Pair-Id=APKAIUCZBIA4LVPAVW3Q&Signature=QKmqmPTAv4CBfFtG1UMXsQHrEcpsygI2pJHd7ryOUIfzIPDZaTEixI9l0UmY8Q-5155UD5ZcP4TmS6eC~0jitUpYmB9p3-FWErlsbjwcntq~NHjUFqmyUzHMw6oy0zz1MmNjC5MHGv4mUCe8s~AUKUuzkpVnDP3bZU0CR-zKtU2Sp7~4cE84XR~cdGE~A8sZfpKq0YFWSPYjokkiM4pEUtmGUNrCzrnd6pgcPMwH~PnyRn~8cTgh1liRPuES4-ER4I3nmhA6WYXDZaIxMYJg9wdCaE1oPCKP9CsFN~VkAtK04SQLoOU9HyxE~3lpl~S1GOwdGJfMgQ9zbYvYs0a9DQ__ |
| Alternate Webpage(s) | http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC307485&blobtype=pdf |
| Alternate Webpage(s) | https://doi.org/10.1093/nar%2F23.23.4924 |
| Journal | Nucleic acids research |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
