Limits...
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.

Show MeSH

Related in: MedlinePlus

The effect of temperature on the cleavage rate of probes with different length and calculated Tm. The numbers in parentheses are the determined Tms. ‘nd’ is not determined. The target and enhancers sequences are, respectively, 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′ and 3′-CAGCCACGGTTACACCG-5′.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1636472&req=5

fig5: The effect of temperature on the cleavage rate of probes with different length and calculated Tm. The numbers in parentheses are the determined Tms. ‘nd’ is not determined. The target and enhancers sequences are, respectively, 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′ and 3′-CAGCCACGGTTACACCG-5′.

Mentions: Figure 5 shows how the cleavage rate of probes with different Tms changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in Figure 5 with calculated Tms of between 14 and 60°C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated Tms of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated Tm or slightly above it. Performing the assay at a temperature slightly higher than the Tm (Figure 5) showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this 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)

The effect of temperature on the cleavage rate of probes with different length and calculated Tm. The numbers in parentheses are the determined Tms. ‘nd’ is not determined. The target and enhancers sequences are, respectively, 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′ and 3′-CAGCCACGGTTACACCG-5′.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: The effect of temperature on the cleavage rate of probes with different length and calculated Tm. The numbers in parentheses are the determined Tms. ‘nd’ is not determined. The target and enhancers sequences are, respectively, 5′-AGTCACAGTCGGTGCCAATGTGGCGGGCAAGGACCGAGTCG-3′ and 3′-CAGCCACGGTTACACCG-5′.
Mentions: Figure 5 shows how the cleavage rate of probes with different Tms changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in Figure 5 with calculated Tms of between 14 and 60°C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated Tms of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated Tm or slightly above it. Performing the assay at a temperature slightly higher than the Tm (Figure 5) showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this 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