RESUMO
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.
Assuntos
Espectrometria de Massas/métodos , Repetições de Microssatélites , Técnicas de Diagnóstico Molecular , Biotecnologia/métodos , Genótipo , Humanos , Perda de Heterozigosidade , Sequências Repetitivas de Ácido Nucleico , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
DNA Viral/sangue , Anticorpos Anti-Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/genética , Humanos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/análise , Nucleotídeos/metabolismo , Análise de Sequência de DNA/métodos , Taq Polimerase/metabolismo , DNA/metabolismo , Primers do DNA , Amplificação de Genes , Reação em Cadeia da PolimeraseRESUMO
The effects of ammonium hydroxide treatment of complexes consisting of biotinylated nucleic acids and immobilized streptavidin were investigated. It was found that incubation of such complexes with ammonium hydroxide at room temperature leads to denaturation of double-stranded DNA molecules, liberating only the complementary nonbiotinylated strand, whereas incubation at elevated temperatures leads to an efficient dissociation of biotin-streptavidin complexes. The introduced procedure is especially suitable as a purification and conditioning format prior to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of DNA from complex enzymatic reactions. This is demonstrated by analysis of polymerase chain reaction (PCR) and sequencing products.
Assuntos
Ácidos Nucleicos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Hidróxido de Amônia , Proteínas de Bactérias , Biotina , DNA/química , Hidróxidos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , EstreptavidinaRESUMO
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.
Assuntos
DNA Viral/análise , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Hepatite B/sangue , Vírus da Hepatite B/genética , HumanosRESUMO
A rapid and accurate detection of ligation products generated in ligase chain reactions (LCR) by using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) is reported. LCR with Pfu DNA ligase was performed with a wild-type template and a template carrying a single point mutation within the Escherichia coli lacI gene as a model system. Starting from about 1 fmol of template DNA the ligation product generated in the positive reactions was analyzed with HPLC and MALDI-TOF-MS, whereby the need of proper sample purification prior to mass spectrometric analysis was demonstrated. A purification procedure with a high potential for automation using streptavidin-coated magnetic particles and ultrafiltration was introduced. Plasmid DNA and short single-stranded oligonucleotides have been used as template. A point mutation could be discriminated from the wild-type template due to the absence or presence of ligation product. This approach allows the rapid-specific detection of template DNA in femtomole amounts and moreover can distinguish between sequence variations in DNA molecules down to point mutations without the need for labeling, gel electrophoresis, membrane transfer, or hybridization procedures.