Röteln (s. S. 5)

Germer, W. D.; Lode, H.; Stickl, H.

doi:10.1007/978-3-642-83160-7_25

Taschenbücher Allgemeinmedizin

1987

DOI: 10.1007/978-3-642-83160-7_25

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Röteln (s. S. 5)

W. D. Germer¹,

H. Lode

H. Stickl³

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2012

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confidence: 99%

Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

Takei¹,

Igarashi²,

Oka³

et al. 2012

ChemBioChem

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Allele-specific polymerase chain reaction (AS-PCR) [1][2][3][4][5][6][7][8][9][10] is one of the most convenient and direct methods for single nucleotide polymorphism (SNP) typing, [11][12][13] and is based on the selective elongation of the primer that matches the template sequence. However, the discrimination of two alleles by allele-specific primers is not always complete because of subtle differences in the thermodynamic stability between matched and mismatched primer-template complexes. Elongation of the primer from the mismatched primer-template complex will eventually match the template to the formerly mismatched primer. Because of the exponential amplification of the analyte DNA by PCR, formation of a matched template for the mismatched primer significantly reduces allele specificity. Although methods to improve allele specificity have been developed by using primer design, [14][15][16][17][18][19][20][21] allele specific blocking [22][23][24] and clamping, [25,26] and engineered polymerase, [27][28][29] a complete solution remains to be devised.Another issue in AS-PCR is the fact that diagnosis with AS-PCR usually relies on the detection of amplified double-stranded DNA (dsDNA). Because dsDNA can be produced from both matched and mismatched primer-template complexes, diagnosis with AS-PCR by detecting dsDNA has some intrinsic ambiguity. To address this second issue, we have used primer labeling [30][31][32] and have reported on a hairpin primer PCR (HP-PCR) method, [33] which monitors the progress of PCR by detecting not the producing dsDNA but the amount of primer consumed (or remaining). In the HP-PCR method, the primers are labeled with a hairpin tag containing a cytosine bulge (Cbulge). The fluorescent molecule 2,7-diamino-1,8-naphthyridine (DANP; Scheme 1 A) bound to the C-bulge and emitted fluorescence at around 430-450 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP. The hairpin structure was unfolded and converted into the double-stranded form by DNA polymerase with concomitant release of the bound DANP from the hairpin tag, resulting in a decrease in the fluorescence at 450 nm. The fluorescence intensity was monitored at 450 nm to quantify consumption of the HP in this HP-PCR method. [34,35] Exploiting this ability of HP-PCR to detect the amount of primer, as compared with conventional PCR, which detects the product dsDNA, we here describe our approach to solve the [a] Dr.

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confidence: 99%

Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

Takei¹,

Igarashi²,

Oka³

et al. 2012

ChemBioChem

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Röteln (s. S. 5)

Cited by 1 publication

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Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

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