A rapid and accurate approach to identify single nucleotide polymorphisms of mitochondrial DNA using MALDI-TOF mass spectrometry

BACKGROUND: Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs. METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis. RESULTS: The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis. CONCLUSIONS: Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immunology, transfusion medicine, the diagnosis of mtDNA mutation related disorders, and the research regarding aging, apoptosis and carcinogenesis based on physiologic and pathogenic alterations of mtDNA for the analysis of large-scale samples, multiple SNPs or rare mtDNA

Prevalence and characterization of murine leukemia virus contamination in human cell lines.

Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%

Detection of XPR-1 expression in the monkey cell line VERO-B4 and the human cell lines MEL-JUSO and NCI-H82 by RT-PCR.

<p>VERO-B4 and NCI-H82 express XPR-1 mRNA in significant amounts (1455 bp), whereas MEL-JUSO lacks the expression of XPR-1 transcripts. The demonstration of the ubiquitously expressed ABL mRNA was used to show the integrity of the mRNA and the cDNA (216 bp).</p

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs). STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing. RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci. CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications

Direct DNA Sanger sequencing chromatograms showing sequence polymorphisms in the human cell lines COLO-677, KYSE-70, HN, and the mouse- derived cell line 32D.

<p>COLO-677 and KYSE-70 contain single sequences of MLV contaminants, whereas HN and 32D show identical chromatograms, indicating that the DNA of the human cell line was contaminated with mouse DNA. A single nucleotide deletion (arrows) results in a sequence shift leading to an overlap of two MLV sequences present in the mouse genomic DNA.</p

Neighbor joining tree of full genomic MLV sequences.

<p>The chart demonstrates the relationship of the complete MLV genomes. The alignment and the tree construction were performed with the MEGA6 software. The investigated sequences produce the same major groups of MLV subtypes as found for the partial sequences. The scale bar represents the number of nucleotide substitutions per site. * Sequences obtained from the NCBI sequence database.</p

Detection of RT activity in human and mouse (ELM-I-1 and PSI-2) cell culture supernatants by PERT assay.

<p>The assay was carried out applying two different buffers containing MgCl₂ or MnCl₂, respectively. The Mn²⁺-buffer was more sensitive in most cases, indicating that the contaminating viruses presumably belong to the Mn²⁺-dependent C-type retroviruses. The CML-T1 cell line was MLV-PCR positive due to contamination of the genomic DNA with mouse derived DNA. The size of the MS2 PCR product is 112 bp.</p

Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay.

<p>Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl₂. The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.</p

PCR-Assays for the sensitive and reliable detection of MLV sequences in cell line DNA.

<p>The upper two panels show the ethidium bromide stained gels of the MLV-specific PCR assays performed with the outer (first panel) and inner primers (second panel). The sizes of the MLV-specific bands are 604–642 bp and 150–174 bp, respectively, depending on the MLV genotype. Some cell lines produce an unspecific band with the outer primers (also seen in some MLV-negative cell lines). The inner primers usually also produce several unspecific bands seen only in MLV-positive cell lines. The cell lines KYSE-30 and NAMALWA are MLV-negative in the assay, whereas the cell lines from the same series or subclones are MLV-positive, respectively. The third panel demonstrates the integrity of the DNAs used for the analyses by amplification of the human ABL gene. The lower panel shows contamination of the genomic DNA with mouse DNA by amplification of mouse specific IAP coding sequences. The PCR produces a double band of approximately 300 bp. The cell line NCEB-1 harbors several mouse-derived chromosomes.</p

Nucleotide sequence alignment of MLV infected cell lines.

<p>The multiple sequence alignment was performed using Vector NTI. The multiple sequence file was then imported to showalign of the EMBOSS program collection. The numbers determine the position from the forward primer. Missing nucleotides are represented by a dash and nucleotides different to the consensus sequence are represented by the respective character. * Cell line sequences originating from the NCBI database.</p