Genosnip: SNP genotyping by MALDI-TOF MS using photocleavable oligonucleotides

Article abstract of DOI:10.1081/NCN-120023038

A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthesised and incorporated within oligonucleotides. After allele-specific primer extension, desalting was performed using genostrep purification plates. Release of the SNP information

Full text of DOI:10.1081/NCN-120023038

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Genosnip: SNP Genotyping by MALDI-TOF MS Using
Photocleavable Oligonucleotides
T. Wenzel a b , T. Elssner a , K. Fahr a , J. Bimmler a , S. Richter a , I. Thomas a & M.
Kostrzewa a
a Bruker Daltonik GmbH , Leipzig, Germany
b Bruker Saxonia Analytik Gmblt , Permoserstrasse 15, Leipzig, D-04318, Germany
Published online: 31 Aug 2006.
To cite this article: T. Wenzel , T. Elssner , K. Fahr , J. Bimmler , S. Richter , I. Thomas & M. Kostrzewa (2003) Genosnip:
SNP Genotyping by MALDI-TOF MS Using Photocleavable Oligonucleotides, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8,
1579-1581, DOI: 10.1081/NCN-120023038
To link to this article: http://dx.doi.org/10.1081/NCN-120023038
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1579–1581, 2003
Genosnip: SNP Genotyping by MALDI-TOF MS
Using Photocleavable Oligonucleotides
*
T. Wenzel, T. Elssner, K. Fahr, J. Bimmler, S. Richter, I. Thomas,
and M. Kostrzewa
Bruker Daltonik GmbH, Leipzig, Germany
ABSTRACT
A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthe-
sised and incorporated within oligonucleotides. After allele-specific primer exten-
sion, desalting was performed using genostrep purification plates. Release of the
SNP information containing part through photocleavage created shortened mole-
cules that are easily accessible for MALDI-TOF analysis. Additionally, incor-
poration of mass modified nucleosides enables flexible design of multiplex
genotyping.
INTRODUCTION
Genotyping SNPs by MALDI-TOF MS is a highly sophisticated method
because of its accuracy, cost-effectiveness, speed and automation capability. Major
drawback of current methods for MALDI SNP analysis is complex sample prepara-
tion. This is obligate because of the strong tendency of larger DNA molecules to
form adducts with positively charged ions.
Here, we present genoSNIP, a novel method for SNP analysis using size reduc-
tion of primer extension products by incorporation of a non-hydrogen bonding
*Correspondence: T. Wenzel, Bruker Saxonia Analytik Gmblt, Permoserstrasse 15, D-04318
Leipzig, Germany; Fax: + 49 341 243 1313; E-mail: tw@bsax.de.
1579
DOI: 10.1081/NCN-120023038
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1580
Wenzel et al.
DNA building-block for phototriggering strand breaks near to the 30-end of the
extension primer.
RESULTS AND DISCUSSION
The large-scale synthesis of the photocleavable b-CE phosphoramidite building-
block (3, [1]) was performed using commercially available o-nitrobenzaldehyde (1). 1
was converted into o-nitrophenyl-3-butenol using allyltrimethylsilane in the presence
of TiCl4. Next, diol 2 was generated by ozonolysis of the previous compound and
reductive workup with NaBH4. Dimethoxytritylation of 2 followed by phosphityl-
ation yielded target compound 3 in excellent yields (overall yield: 60%).
To genotype SNPs by genoSNIP genomic regions containing polymorphic sites
are amplified by PCR. The PCR products are used as templates for primer extension
reactions which generate allele specific products. The primers used in the extension
reaction were biotinylated at the 50-end and they did contain the photocleavable
building-block 3 close to the 30-end. After extension reaction, purification was done
by genostrep, a purification system based on streptavidin coated microtiterplates.
(i) 1M TiCl4=CH2Cl2, CH2 ¼ CHCH2Si(CH3)3, H2O; (ii) O3=MeOH, NaBH4, H2O;
(iii) (MeO)2TrCl=pyridine=TEA=DMAP; (iv) [(CH3)2CH]2NP(Cl)OCH2CH2CN=DIPEA=
CH2Cl2.
Purified extension products were photochemically cleaved releasing shortened
DNA molecules as 50-monophosphates.
Figure 1. SNP genotyping of three different risk factors (factor V Leiden G 1691A, Pro-
thrombin G20210A, MTHFR C677 T) using the genoSNIP assay (a,c); singleplex analysis
of MTHFR (a), singleplex analysis of mass increased MTHFR (b), triplex analysis (c).

SNP Genotyping by MALDI-TOF MS
1581
Here, we present SNP genotyping by genoSNIP=genostrep using assays for sev-
eral common genetic risk factors (Prothrombin, Factor V Leiden, MTHFR).
As the primer extension products of MTHFR (M ¼ 1592 Da) and factor V
Leiden (M ¼ 1592 Da) show the same molecular weight, we introduced 20-OMe-
guanosine close to the photocleavable building-block of MTHFR increasing the
weight by 30 Da.
The incorporation of mass modified nucleoside within DNA enables multiplex-
ing. Combination of genoSNIP=genostrep purification empowers rapid and cost-
effective genotyping which is easy to automate.
REFERENCE
1. Ordoukhanian, P.; Taylor, J.S. J. Am. Chem. Soc. 1995, 117 (37), 9570–9571.