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

STONEBERG HOLT, Sierra Dawn, Lucie HOROVÁ and Petr BUREŠ. Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR Green II staining. Taxon. Vienna: International Assoc. for Plant Taxonomy, 2007, vol. 56, No 2, p. 509-515. ISSN 0040-0262.

Basic information

• Original name: Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR Green II staining
• Name in Czech: Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR Green II staining
• Authors: STONEBERG HOLT, Sierra Dawn (840 United States of America), Lucie HOROVÁ (203 Czech Republic) and Petr BUREŠ (203 Czech Republic, guarantor).
• Edition: Taxon, Vienna, International Assoc. for Plant Taxonomy, 2007, 0040-0262.

Other information

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: 10600 1.6 Biological sciences
• Country of publisher: Austria
• Confidentiality degree: is not subject to a state or trade secret
• Impact factor: Impact factor: 2.524
• RIV identification code: RIV/00216224:14310/07:00022322
• Organization unit: Faculty of Science
• UT WoS: 000244825300015
• Keywords in English: chemical cleavage of mismatch (CCM); cpDNA; heteroduplex analysis (HA); intraspecific sequence studies; phylogeography; SYBR Green II staining
• Tags: cpDNA, heteroduplex analysis (HA), intraspecific sequence studies, phylogeography, SYBR Green II staining
• Tags: International impact, Reviewed

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. 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.

Abstract (in Czech)

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.

Links

• LC06073, research and development project: Name: Centrum pro výzkum biodiverzity

Investor: Ministry of Education, Youth and Sports of the CR, Biodiversity Research Center

• MSM0021622416, plan (intention): Name: Diverzita biotických společenstev a populací: kauzální analýza variability v prostoru a čase

Investor: Ministry of Education, Youth and Sports of the CR, Diversity of Biotic Communities and Populations: Causal Analysis of variation in space and time