Novel Propargylamine-Linked Nucleosides for High Throughput SNP Genotyping by Maldi-Tof MS

Wenzel, Thomas J.; Fröhlich, Thomas; Strassburger, Kathrin; Richter, Susann; Bimmler, Jacqueline; Franke, Constance; Thomas, I. L.; Kostrzewa, Markus

doi:10.1081/ncn-100002451

Cited by 7 publications

(3 citation statements)

References 7 publications

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“…As a consequence, conventional buffers are used for the molecular biology, and are not removed prior to analysis. A propargylamine-linked nucleoside (Wenzel et al, 2001) is placed on the second position from the 3 0 -terminus of the extension primer, and is used to attach different trialkylammoniumalkyryl-N-hydroxysuccinimidyl esters (Bartlet-Jones et al, 1994). A quaternary ammonium group defines a single positive charge, and, by using different alkyl chain lengths, the masses of the alleles of SNPs can be shifted to increase the degrees of freedom in designing multiplexes.…”

Section: The Good Assaymentioning

confidence: 99%

Genotyping single nucleotide polymorphisms by mass spectrometry

Tost

Gut

2002

Mass Spectrometry Reviews

120

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In the last decade, the demand for high-throughput DNA analysis methods has dramatically increased, mainly due to the advent of the human genome sequencing project that is now nearing completion. Even though mass spectrometry did not contribute to that project, it is clear that it will have an important role in the post-genome sequencing era, in genomics and proteomics. In genomics, mainly matrix-assisted laser desorption/ionization (MALDI) mass spectrometry will contribute to large-scale single nucleotide polymorphism (SNP) genotyping projects. Here, the development and history of DNA analysis by mass spectrometry is reviewed and put into the context with the requirements of genomics. All major contributions to the field and their status and limitations are described in detail.

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Section: The Good Assaymentioning

confidence: 99%

Genotyping single nucleotide polymorphisms by mass spectrometry

Tost

Gut

2002

Mass Spectrometry Reviews

120

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“…Extension primers for positive charge-tagging carry an aminopropargyl modification on the second base from the 3 0 -end. 19,20 The amino functionality is used for attaching a positive charge-tag (6-trimethylammoniumhexyryl-N-hydroxysuccinimidyl ester) according to Gut et al 15 and Bartlet-Jones et al 21 U NH2 is converted into U CT and A NH2 into A CT . The primers containing the amino functionality were dissolved in 1% TE-buffer at 500 pmol/mL.…”

Section: Positive Charge-taggingmentioning

confidence: 99%

Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix‐assisted laser desorption/ionization mass spectrometry

Sauer

Gut

2003

Rapid Comm Mass Spectrometry

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Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or a-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease. Copyright # 2003 John Wiley & Sons, Ltd.Genotyping of single nucleotide polymorphisms (SNPs) is considered the ideal strategy for dissecting complex traits in association studies and linkage disequilibrium mapping. 1Determining the relevance of SNPs for certain phenotypes will require comparative studies of thousands of affected and unaffected individuals.2 The analysis of SNPs could also find its application in pharmacogenetics where the choice of drugs could be customized for individual patientspecific responses. 3 Furthermore, traceability of food could be performed using SNP markers. Many methods, such as DNA microarrays, gel-based and plate-reader based assays for SNP genotyping, have been introduced but none of these compete with the speed and resolution of mass spectrometry. [4][5][6] In particular, matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) has revolutionized the instrumental analysis of biomolecules. 7,8 Unfortunately, most of the methods for the analysis of DNA, like genotyping SNPs by MALDI, require stringent purification procedures that are expensive and cumberso...

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“…A further impact concerns the incorporation of charge tags into nucleic acids (DNA and RNA) in order to enhance their detectability in MALDI-TOF mass spectrometry. It has been shown that the incorporation of already one charge into a DNA molecule enhances the sensitivity by which it can be detected by a factor of 100 (Wenzel et al, 2001;Gut and Beck, 1995). This result has recently found application in the development of a novel procedure for efficient genotyping of single nucleotide polymorphism ('GOOD' assay) (Sauer et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Oligonucleotides Incorporating 7-(Aminoalkynyl)-7-deaza-2‘-deoxyguanosines: Duplex Stability and Phosphodiester Hydrolysis by Exonucleases

et al. 2002

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Oligonucleotides containing 7-(omega-aminoalkyn-1-yl)-7-deaza-2'-deoxyguanosines (1a-c) were investigated regarding their thermal stability (T(m) values) as well as their phosphodiester hydrolysis catalyzed by exonucleases. Those derivatives are suitable for the labeling of nucleic acid constituents as well as for the postlabeling of DNA. For this, the phosphoramidites 7a,c (obtained from the nucleoside 1a,b), protected by an isobutyryl group at the 2-amino group and a phthaloyl residue at the side-chain amino function, were synthesized. Using compounds 7a,c together with the phosphoramidite of 1c in solid-phase synthesis, a series of self-complementary and non-self-complementary oligonucleotides were prepared and characterized by MALDI-TOF mass spectrometry. A comparison of the T(m) values of the modified oligomers shows that the thermal stability of the duplexes decreases with the length of the nucleobase 7-(omega-aminoalkyn-1-yl) side chain. Exonucleolytic cleavage of oligonucleotide single strands incorporating either the 7-(3-aminopropyn-1-yl)- or the 7-(4-aminobutyn-1-yl)-substituted nucleosides 1a or 1b, respectively, reveals that 3' --> 5' specific snake venom phosphodiesterase liberates 1a 5'-monophosphate but not the methylene-extended 1b 5'-monophosphate. On the contrary, the 5' --> 3' specific bovine spleen exonuclease is able to cleave off single 1a and 1b 3'-monophosphate residues; its action is, however, terminated in the case of oligonucleotides containing two consecutive 1a or 1b nucleotide units.

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Novel Propargylamine-Linked Nucleosides for High Throughput SNP Genotyping by Maldi-Tof MS

Cited by 7 publications

References 7 publications

Genotyping single nucleotide polymorphisms by mass spectrometry

Genotyping single nucleotide polymorphisms by mass spectrometry

Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix‐assisted laser desorption/ionization mass spectrometry

Oligonucleotides Incorporating 7-(Aminoalkynyl)-7-deaza-2‘-deoxyguanosines: Duplex Stability and Phosphodiester Hydrolysis by Exonucleases

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