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A novel endonuclease IV post-PCR genotyping system.

Kutyavin IV, Milesi D, Belousov Y, Podyminogin M, Vorobiev A, Gorn V, Lukhtanov EA, Vermeulen NM, Mahoney W - Nucleic Acids Res. (2006)

Bottom Line: Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA.In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated.High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

View Article: PubMed Central - PubMed

Affiliation: Nanogen, 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA.

ABSTRACT
Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

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Related in: MedlinePlus

(A) First double strand DNA lesion where ‘a’ represents an abasic 2′-deoxyribose residue and ‘p’ is a phosphate group. (B) Second DNA lesion, formed by spontaneous or enzymatic cleavage of the phosphodiester bond between the 3′-hydroxyl group of the 2′-deoxyribose (abasic site) and the nearest nucleotide of the DNA strand. ‘s’ represents several products of 2′-deoxyribose degradation. (C) Artificial abasic lesion generated by the novel probe and enhancer. Arrows indicated the specific cleavage by AP endonuclease.
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fig1: (A) First double strand DNA lesion where ‘a’ represents an abasic 2′-deoxyribose residue and ‘p’ is a phosphate group. (B) Second DNA lesion, formed by spontaneous or enzymatic cleavage of the phosphodiester bond between the 3′-hydroxyl group of the 2′-deoxyribose (abasic site) and the nearest nucleotide of the DNA strand. ‘s’ represents several products of 2′-deoxyribose degradation. (C) Artificial abasic lesion generated by the novel probe and enhancer. Arrows indicated the specific cleavage by AP endonuclease.

Mentions: Here we describe a novel endonuclease IV (Endo IV) assay substrate (Figure 1C) that mimics the abasic lesions that normally occur in double-stranded DNA (Figure 1A and B). The first lesion (Figure 1A) is a typical abasic (apurinic or apyrimidinic site generated by spontaneous or enzymatic loss of a nucleic acid base. The second lesion (Figure 1B) is an atypical abasic site appearing as a result of inherent instability of the 3′-phosphodiester bond in abasic deoxyribose in lesion 1 or its cleavage by a Class I AP endonuclease. As shown by the arrow, AP endonucleases cleave the phosphodiester linkage at the abasic sites. In the novel Endo IV assay a short probe with a fluorophore linked through a phosphate at the 3′ end and a short enhancer oligonucleotide generate an artificial, lesion 2 type Endo IV substrate. This arrangement allows the specific and efficient cleavage of the phosphodiester bond of the probe by Endo IV and release of fluorescent dye (Figure 1C). The addition of a quencher at the 5′ end of the probe allows quenching of the fluorescence in the uncleaved probe. The specific cleavage of the phosphate bond and the generation of fluorescence is the basis of the new assay.


A novel endonuclease IV post-PCR genotyping system.

Kutyavin IV, Milesi D, Belousov Y, Podyminogin M, Vorobiev A, Gorn V, Lukhtanov EA, Vermeulen NM, Mahoney W - Nucleic Acids Res. (2006)

(A) First double strand DNA lesion where ‘a’ represents an abasic 2′-deoxyribose residue and ‘p’ is a phosphate group. (B) Second DNA lesion, formed by spontaneous or enzymatic cleavage of the phosphodiester bond between the 3′-hydroxyl group of the 2′-deoxyribose (abasic site) and the nearest nucleotide of the DNA strand. ‘s’ represents several products of 2′-deoxyribose degradation. (C) Artificial abasic lesion generated by the novel probe and enhancer. Arrows indicated the specific cleavage by AP endonuclease.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636472&req=5

fig1: (A) First double strand DNA lesion where ‘a’ represents an abasic 2′-deoxyribose residue and ‘p’ is a phosphate group. (B) Second DNA lesion, formed by spontaneous or enzymatic cleavage of the phosphodiester bond between the 3′-hydroxyl group of the 2′-deoxyribose (abasic site) and the nearest nucleotide of the DNA strand. ‘s’ represents several products of 2′-deoxyribose degradation. (C) Artificial abasic lesion generated by the novel probe and enhancer. Arrows indicated the specific cleavage by AP endonuclease.
Mentions: Here we describe a novel endonuclease IV (Endo IV) assay substrate (Figure 1C) that mimics the abasic lesions that normally occur in double-stranded DNA (Figure 1A and B). The first lesion (Figure 1A) is a typical abasic (apurinic or apyrimidinic site generated by spontaneous or enzymatic loss of a nucleic acid base. The second lesion (Figure 1B) is an atypical abasic site appearing as a result of inherent instability of the 3′-phosphodiester bond in abasic deoxyribose in lesion 1 or its cleavage by a Class I AP endonuclease. As shown by the arrow, AP endonucleases cleave the phosphodiester linkage at the abasic sites. In the novel Endo IV assay a short probe with a fluorophore linked through a phosphate at the 3′ end and a short enhancer oligonucleotide generate an artificial, lesion 2 type Endo IV substrate. This arrangement allows the specific and efficient cleavage of the phosphodiester bond of the probe by Endo IV and release of fluorescent dye (Figure 1C). The addition of a quencher at the 5′ end of the probe allows quenching of the fluorescence in the uncleaved probe. The specific cleavage of the phosphate bond and the generation of fluorescence is the basis of the new assay.

Bottom Line: Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA.In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated.High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

View Article: PubMed Central - PubMed

Affiliation: Nanogen, 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA.

ABSTRACT
Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

Show MeSH
Related in: MedlinePlus