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Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR® Green II staining

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Abstract:

Studies of organelle DNA sequence at low taxonomic levels present a researcher with specific experimental design challenges. Such studies require sampling numerous individuals, many of which share identical haplotypes. The process is often limited by the time and money required for DNA sequencing. A number of screening techniques have been devised to combat this limitation, but many have not found wide-spread use in botany. An ideal screening technique would be fast, easy, safe, inexpensive, detect 100% of mutations, be suitable for DNA fragments of about 1 kb, and not require complex equipment. This paper compares the heteroduplex analysis (HA) and chemical cleavage of mismatch (CCM) methods for detecting known point mutations and a deletion in a 1 kb region of non-coding cpDNA from the Poa pratensis agg. A new CCM visualization method, staining with SYBR® Green II, was tested.

Keywords: CHEMICAL CLEAVAGE OF MISMATCH (CCM); CPDNA; HETERODUPLEX ANALYSIS (HA); INTRASPECIFIC SEQUENCE STUDIES; PHYLOGEOGRAPHY; SYBR GREEN II STAINING

Document Type: Research Article

Affiliations: 1: Institute of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, Brno 611 37, Czech Republic; Box 37, Hinsdale, Montana 59241, U.S.A. 2: Institute of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, Brno 611 37, Czech Republic

Publication date: 2007-05-01

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