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PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning

Saunders, Robert D. C. and Glover, David M. and Ashburner, Michael and Siden-Kiamos, Inga and Louis, Christos and Monastirioti, Maria and Savakis, Charalambos and Kafatos, Fotis (1989) PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning. Nucleic Acids Research, 17 (22). pp. 9027-9037. ISSN 0305-1048. PMCID PMC335111. https://resolver.caltech.edu/CaltechAUTHORS:20200807-170351295

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Abstract

A novel alternative to microcloning for the production of region specific chromosomal DNA is described. In this method, ‘microamplification’, single bands are dissected from polytene chromosomes and digested with Sau3A. Oligonucleotide adaptors are ligated to these fragments to provide convenient priming sites for polymerase chain reaction amplification. In this way, as much as 1μg of DNA can be amplified from a single band. Probes made from PCR amplified DNA from two such dissections have been used to probe cloned DNA form a 100kb chromosome walk. Whereas conventional microcloning has generated cloned EcoRI fragments corresponding to 3–4kb of the walk, the PCR probes cover greater than 90% of this chromosomal region. Thus microamplification is significantly more effective than microcloning in providing probes for establishing chromosomal walks.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/nar/17.22.9027DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC335111/PubMed CentralArticle
ORCID:
AuthorORCID
Glover, David M.0000-0003-0956-0103
Additional Information:© 1989 IRL Press Limited. Creative Commons Attribution Non-Commercial licence (CC BY-NC). Received September 4, 1989; Accepted October 10, 1989. This work was supported by an Operation contract of the Stimulation Programme of the European Economic Community number ST2P-0477-C(TT). The early work on the asp region was supported by an MRC project grant to RDCS and DMG. DMG also wishes to acknowledge a career development award from the Cancer Research Campaign. We wish to acknowledge the following colleagues: P.F.R. Little for much helpful discussion, and pointing out the useful characteristics of DpnI, C.Gonzalez for help with the cytogenetics of the asp region, and H. Jaeckle for advice concerning microcloning, and A. Cheshire for help with photography.
Funders:
Funding AgencyGrant Number
European UnionST2P-0477-C
Medical Research Council (UK)UNSPECIFIED
Cancer Research CampaignUNSPECIFIED
Issue or Number:22
PubMed Central ID:PMC335111
Record Number:CaltechAUTHORS:20200807-170351295
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200807-170351295
Official Citation:Robert D.C. Saunders, David M. Glover, Michael Ashburner, Inga Siden-Kiamos, Christos Louis, Maria Monastirioti, Charalambos Savakis, Fotis Kafatos, PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning, Nucleic Acids Research, Volume 17, Issue 22, 25 November 1989, Pages 9027–9037, https://doi.org/10.1093/nar/17.22.9027
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104845
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:11 Aug 2020 19:57
Last Modified:11 Aug 2020 19:57

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