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Chemical cleavage reactions of DNA on solid support: application in mutation detection.

Bui CT, Lambrinakos A, Babon JJ, Cotton RG - BMC Chem Biol (2003)

Bottom Line: BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step.The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps.CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genomic Disorders Research Centre (the University of Melbourne) 7th Floor, Daly Wing, St, Vincent's Hospital, 35 Victoria parade, Fitzroy, Vic, 3065, Australia. chinhbui@mail.medstv.unimelb.edu.au

ABSTRACT
BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. RESULTS: DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

No MeSH data available.


Chemical Cleavage of Mismatch. * DNA strand was labeled with FAM or HEX at 5' and 3' ends respectively. Heteroduplex was formed by mixing equal amounts of wild-type and mutant DNA. Mismatched T.C on the heteroduplex was modified by KMnO4 and hydroylamine and then cleaved by piperidine. A denaturing gel displays two cleavage fragments derived from both strands. Note: When only one of two reagents (either KMnO4 or hydroxylamine) was applied on T.C mismatch, only one labeled cleavage fragment was detected by gel-electrophoresis – See Fig. 2 &3).
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Figure 1: Chemical Cleavage of Mismatch. * DNA strand was labeled with FAM or HEX at 5' and 3' ends respectively. Heteroduplex was formed by mixing equal amounts of wild-type and mutant DNA. Mismatched T.C on the heteroduplex was modified by KMnO4 and hydroylamine and then cleaved by piperidine. A denaturing gel displays two cleavage fragments derived from both strands. Note: When only one of two reagents (either KMnO4 or hydroxylamine) was applied on T.C mismatch, only one labeled cleavage fragment was detected by gel-electrophoresis – See Fig. 2 &3).

Mentions: The chemical cleavage of mismatch has been widely used as a diagnostic tool for detection of mutations in many inherited diseases [1,9]. The method was based on the formation of heteroduplexes DNA containing a single base mismatch by mixing equal amounts of two homoduplexes (wild-type and mutant) DNA. At the mismatched site, nucleotide bases are extra-helical and susceptible to chemical modification reactions [10]. Therefore, KMnO4 and hydroxylamine have been employed in the CCM method to modify the mismatched thymine and cytosine respectively. The resulting modified DNA is then treated with piperidine before being loaded on to a sequencing polyacrylamide gel for fragment analysis (Figure 1). This process is time-consuming as the reactions are carried out in solution phases and the products require exhaustive purification procedures after each reaction step.


Chemical cleavage reactions of DNA on solid support: application in mutation detection.

Bui CT, Lambrinakos A, Babon JJ, Cotton RG - BMC Chem Biol (2003)

Chemical Cleavage of Mismatch. * DNA strand was labeled with FAM or HEX at 5' and 3' ends respectively. Heteroduplex was formed by mixing equal amounts of wild-type and mutant DNA. Mismatched T.C on the heteroduplex was modified by KMnO4 and hydroylamine and then cleaved by piperidine. A denaturing gel displays two cleavage fragments derived from both strands. Note: When only one of two reagents (either KMnO4 or hydroxylamine) was applied on T.C mismatch, only one labeled cleavage fragment was detected by gel-electrophoresis – See Fig. 2 &3).
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Related In: Results  -  Collection

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Figure 1: Chemical Cleavage of Mismatch. * DNA strand was labeled with FAM or HEX at 5' and 3' ends respectively. Heteroduplex was formed by mixing equal amounts of wild-type and mutant DNA. Mismatched T.C on the heteroduplex was modified by KMnO4 and hydroylamine and then cleaved by piperidine. A denaturing gel displays two cleavage fragments derived from both strands. Note: When only one of two reagents (either KMnO4 or hydroxylamine) was applied on T.C mismatch, only one labeled cleavage fragment was detected by gel-electrophoresis – See Fig. 2 &3).
Mentions: The chemical cleavage of mismatch has been widely used as a diagnostic tool for detection of mutations in many inherited diseases [1,9]. The method was based on the formation of heteroduplexes DNA containing a single base mismatch by mixing equal amounts of two homoduplexes (wild-type and mutant) DNA. At the mismatched site, nucleotide bases are extra-helical and susceptible to chemical modification reactions [10]. Therefore, KMnO4 and hydroxylamine have been employed in the CCM method to modify the mismatched thymine and cytosine respectively. The resulting modified DNA is then treated with piperidine before being loaded on to a sequencing polyacrylamide gel for fragment analysis (Figure 1). This process is time-consuming as the reactions are carried out in solution phases and the products require exhaustive purification procedures after each reaction step.

Bottom Line: BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step.The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps.CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genomic Disorders Research Centre (the University of Melbourne) 7th Floor, Daly Wing, St, Vincent's Hospital, 35 Victoria parade, Fitzroy, Vic, 3065, Australia. chinhbui@mail.medstv.unimelb.edu.au

ABSTRACT
BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. RESULTS: DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

No MeSH data available.