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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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The comparison of the change in relative signal fluorescence of match and different mismatches at different positions in a 14mer probe in an Endo IV assay run at 60°C. The probe sequence of the matched probe and target sequence are, respectively, 5′-Q-ACTCGGTCCTTGCC-FL-3′ and 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′. All the complementary sequences indicating the different mismatches are shown in the Supplementary Data. NTC is the no template control.
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fig7: The comparison of the change in relative signal fluorescence of match and different mismatches at different positions in a 14mer probe in an Endo IV assay run at 60°C. The probe sequence of the matched probe and target sequence are, respectively, 5′-Q-ACTCGGTCCTTGCC-FL-3′ and 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′. All the complementary sequences indicating the different mismatches are shown in the Supplementary Data. NTC is the no template control.

Mentions: The ability of the Endo IV assay to discriminate mismatches at different positions of a 14mer probe is illustrated in Figure 7. Mismatches were introduced in the probe one at a time, from positions 1 to 8. The Endo IV assay shows excellent specificity for the different mismatches from base 1 to base 6 with large match/mismatch signal ratios. As shown in Figure 7, the mismatch signal is often about the same as that of the NTC or slightly higher. The exceptions are the difficult 4-G/T- and 8-T/G-mismatches (24) where match/mismatch ratios of ∼16 and 7 were observed, respectively. These ratios are quite adequate to differentiate matched and mismatched sequences. It appears (data not shown) that the discrimination in positions 1 and 2 is largely determined by substrate requirements while discrimination in other positions is determined by thermodynamic considerations. Experiments with a large number of assays have also indicated that, with 10mer to 12mer probes, satisfactory discrimination is observed with the mismatch position in all except the last three bases at the 5′ end.


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)

The comparison of the change in relative signal fluorescence of match and different mismatches at different positions in a 14mer probe in an Endo IV assay run at 60°C. The probe sequence of the matched probe and target sequence are, respectively, 5′-Q-ACTCGGTCCTTGCC-FL-3′ and 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′. All the complementary sequences indicating the different mismatches are shown in the Supplementary Data. NTC is the no template control.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: The comparison of the change in relative signal fluorescence of match and different mismatches at different positions in a 14mer probe in an Endo IV assay run at 60°C. The probe sequence of the matched probe and target sequence are, respectively, 5′-Q-ACTCGGTCCTTGCC-FL-3′ and 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′. All the complementary sequences indicating the different mismatches are shown in the Supplementary Data. NTC is the no template control.
Mentions: The ability of the Endo IV assay to discriminate mismatches at different positions of a 14mer probe is illustrated in Figure 7. Mismatches were introduced in the probe one at a time, from positions 1 to 8. The Endo IV assay shows excellent specificity for the different mismatches from base 1 to base 6 with large match/mismatch signal ratios. As shown in Figure 7, the mismatch signal is often about the same as that of the NTC or slightly higher. The exceptions are the difficult 4-G/T- and 8-T/G-mismatches (24) where match/mismatch ratios of ∼16 and 7 were observed, respectively. These ratios are quite adequate to differentiate matched and mismatched sequences. It appears (data not shown) that the discrimination in positions 1 and 2 is largely determined by substrate requirements while discrimination in other positions is determined by thermodynamic considerations. Experiments with a large number of assays have also indicated that, with 10mer to 12mer probes, satisfactory discrimination is observed with the mismatch position in all except the last three bases at the 5′ end.

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