Christian Jurinke

MALDI-TOF mass spectrometry : A versatile tool for high-performance DNA analysis

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the anlaysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.

The use of MassARRAY technology for high throughput genotyping.

This chapter will explore the role of mass spectrometry (MS) as a detection method for genotyping applications and will illustrate how MS evolved from an expert-user-technology to a routine laboratory method in biological sciences. The main focus will be time-of-flight (TOF) based devices and their use for analyzing single-nucleotide-polymorphisms (SNPs, pronounced snips). The first section will describe the evolution of the use of MS in the field of bioanalytical sciences and the protocols used during the early days of bioanalytical MALDI TOF mass spectrometry. The second section will provide an overview on intraspecies sequence diversity and the nature and importance of SNPs for the genomic sciences. This is followed by an exploration of the special and advantageous features of mass spectrometry as the key technology in modern bioanalytical sciences in the third chapter. Finally, the fourth section will describe the MassARRAY technology as an advanced system for automated high-throughput analysis of SNPs.

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 microliters serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a 32P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.

Detection of hepatitis B virus DNA in serum samples via nested PCR and MALDI-TOF mass spectrometry

In a blind study, nested polymerase chain reaction (PCR) was performed with control DNA and DNA preparations from serum samples of six patients. The detection limit was determined to be 100 molecules of template in 1 ml of serum. Hepatitis B virus (HBV) related products of nested PCR were purified by ultrafiltration and immobilisation on streptavidin coated magnetic beads. The immobilized PCR products were denatured from the beads and analyzed via matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. The results of MALDI-TOF MS analysis were in agreement with the results obtained by polyacrylamide gel electrophoresis (PAGE) and with the data obtained by serological analysis. The detection strategy introduced here has a high potential for automation and represents a fast and reliable method of detection for HBV DNA in serum without the need for time consuming gel electrophoresis and labeling or hybridization procedures.

Direct detection of synthetic and biologically generated double-stranded DNA by MALDI-TOF MS

Abstract A synthetic 50-mer DNA was mixed at room temperature with a non-complementary or a complementary 27-mer and analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. The former resulted in negligible signals from a 27-/50- mer heterodimer, for the latter this was a dominant peak, consistent with maintaining Watson-Crick interactions in the gas phase. Double stranded DNA derived from an enzymatic digestion of a 252-base-pair polymerase chain reaction product when prepared at room temperature yielded very little specific double stranded DNA, but sample preparation at reduced temperature resulted in spectra dominated by the expected heterodimer signals with no random (i.e. non-Watson-Crick) dimers. As a DNA diagnostics genotyping application, apolipoprotein-E alleles were easily distinguished considering only the masses of their double stranded DNA digest fragments.

Combined amplification and sequencing in a single reaction using two DNA polymerases with differential incorporation rates for dideoxynucleotides

We describe an approach, which combines the process of DNA amplification and sequence determination by using a pair of primers and two DNA polymerases with different incorporation rates for dideoxynucleotides. The process of target sequence amplification is carried out by the DNA polymerase with a low dideoxynucleotide incorporation rate while its polymerase counterpart with a high incorporation rate generates a sequence ladder. The needs for separate amplification via polymerase chain reaction (PCR) or cloning into plasmids including the respective purification steps therefore can be avoided. In addition, the use of dye terminator chemistry enables the simultaneous generation of forward and reverse sequence ladders, which can be separated based on the streptavidin-biotin system when one amplification primer is biotinylated.

Dna Analysis by Mass Spectrometry: Applications in Dna Sequencing And Dna Diagnostics

Abstract Use of mass spectrometry to detect PCR amplified DNA from individuals infected with hepatitis B virus, to derive DNA sequence information through exo-nuclease based degradation or Sanger sequencing and a new format (PROBE) for the efficient determination of mutations is described.

Microsatellites: perspectives and potentials of mass spectrometric analysis

Mass spectrometry is a powerful analytical tool in biotechnology. The ´soft´ ionization and desorption technologies matrix-assisted laser desorption/ionization and electrospray ionization have enabled mass spectrometric analysis of large biomolecules, such as proteins and nucleic acid amplification products, and paved the way for mass spectrometry to become a leading technology in current genomics and proteomics efforts. Large-scale analysis of single nucleotide polymorphisms by mass spectrometry has been commercially established. This article reviews applications of mass spectrometry for microsatellite analysis. Features and capabilities of the two most prominent techniques, matrix assisted laser desorption/ionization and electrospray-ionization mass spectrometry, are compared and their potential to address the limitations of conventional microsatellite analysis based on comparison of gel electrophoretic mobilities is explored.

Mass Spectrometric DNA Diagnostics

Abstract Mass spectrometry was employed to analyze gene expression of cancer cells by RT-PCR, to determine polymorphisms within short tandem repeat regions of the human genome and to derive sequence information from diagnostic PCR products by solid-phase Sanger sequencing.

Sequence Analysis of Nucleic Acids by Mass Spectrometry

The most widely used methods for DNA sequence determination are enzymatic sequencing reactions (Sanger, 1977) and the base-specific chemical cleavage (Maxam, 1977). Visualization of the obtained sequence ladder is based on the detection of radioisotopic, chemiluminescent or fluorescent labels after the fragment mixture has been separated by gel electrophoresis.