Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR Green II staining
| Authors | |
|---|---|
| Year of publication | 2007 |
| Type | Article in Periodical |
| Magazine / Source | Taxon |
| MU Faculty or unit | |
| Citation | |
| Field | Botany |
| Keywords | chemical cleavage of mismatch (CCM); cpDNA; heteroduplex analysis (HA); intraspecific sequence studies; phylogeography; SYBR Green II staining |
| Description | 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. CCM is a powerful tool for discovering different haplotypes in DNA sequence studies at low taxonomic levels. It is quicker and less expensive than sequencing each sample. Compared to HA, CCM is much more sensitive and delivers results in a shorter time, although it is more expensive and considerably more labor intensive. Staining with SYBR Green II allows CCM to be effectively implemented in laboratories with limited access to automated sequencing equipment. |
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