Lsv

Fig. 1 Base-specific cleavage by MALDI-TOF mass spectrometry. A single-stranded copy of a PCR-amplified target sequence is generated by T7-mediated in vitro transcription and cleaved in four reactions at positions corresponding to each of the four bases. RNA of the forward strand is cleaved at U or C. An A- and G-specific cleavage of the template sequence is facilitated by U or C cleavage of the reverse RNA strand. MALDI-TOF acquisition of spectra of each of the cleavage reactions is followed by comparison to reference sequence derived in silico cleavage pattern.

of the 2'-hydroxy group on the polarization of the N-glycosidic bond of the protonated base.

Figure 1 illustrates this robust biochemical scheme for high-throughput differential sequencing. Two PCR reactions of the DNA region of interest introduce a T7-promotor in the forward strand as well in the reverse strand of the amplification product. PCR is followed by shrimp alkaline phosphatase (SAP) treatment to dephos-phorylate any unincorporated desoxy-NTPs. RNA poly-merase, ribonucleotide, and nuclease-resistant nucleotides are added to the mixture. In vitro transcription generates single-stranded RNA and facilitates further amplification. The RNA is subject to four base-specific cleavage reactions corresponding to each of the four bases. Reactions are driven to completion. This reduces the RNA target to a specific set of RNA compomers in each of the reactions. Analytes are desalted by addition of ionexchange resin, conditioned, and identified in a single MALDI-TOF MS measurement per reaction. Four se quence-specific mass signal patterns are generated. All cleavage products are consistent with 5'-OH and 3'-phosphate groups, except for the 3'-fragment of the full-length transcript possessing a 3'-OH group.

The experimental set of compomers is used to reconstruct the sequence by cross-comparing the information of the four cleavages to the in silico cleavage pattern of the known reference sequence. Deviations of the pattern indicate sequence changes (Fig. 1).

Figure 2 shows base-specific, cleavage-mediated discovery of a [C/T] sequence change in a 500-bp DNA region of interest. The target region is analyzed by C- and T-specific cleavage of the forward, as well as the reverse strand—equivalent to four base-specific cleavage reactions. Overlays show spectra of the wild-type as well as heterozygous and homozygous mutant samples.

For the wild-type sample [C/C], mass signals of all cleavage reactions can unambiguously be identified based on the reference sequence derived in silico cleavage

Fig. 2 Identification of a single nucleotide polymorphism by MALDI-TOF MS analysis. Panels A-D show the four base-specific cleavage reactions. Each spectrum reveals the changes resulting from the substitution of a C wt-allele for a T mutant-allele. Spectra of all three different genotypes are overlaid. Changes are indicated by arrows. (A) T-specific cleavage of the forward transcript. (B) C-specific cleavage of the forward transcript. (C) T-specific cleavage of the reverse transcript. (D) C-specific cleavage of the reverse transcript. Spectra are shown covering all detected signals. All signals affected by the sequence change are indicated.

Fig. 2 Identification of a single nucleotide polymorphism by MALDI-TOF MS analysis. Panels A-D show the four base-specific cleavage reactions. Each spectrum reveals the changes resulting from the substitution of a C wt-allele for a T mutant-allele. Spectra of all three different genotypes are overlaid. Changes are indicated by arrows. (A) T-specific cleavage of the forward transcript. (B) C-specific cleavage of the forward transcript. (C) T-specific cleavage of the reverse transcript. (D) C-specific cleavage of the reverse transcript. Spectra are shown covering all detected signals. All signals affected by the sequence change are indicated.

pattern. Deviations from the in silico pattern, detected as a mass shift, the absence of an existing peak, or the appearance of an additional signal, lead to the identification of the sequence variation.

In the T-specific cleavage reaction of the forward transcript, a sequence change from C to T at position 371 of the target region introduces a new cleavage site and splits the 15-bp wild-type fragment into a 12- and a 3-bp fragment. For the mutant [T/T] sample, this results in the disappearance of a mass signal at 4830.0 Da and the appearance of mutant-specific mass signals at 3850 and 1015.6 Da (signal not shown). Spectra containing all of the signals correspond to the heterozygous sample [C/T].

The C-specific cleavage reaction on the forward strand confirms the observation of the identified [C/T] substitution. A cleavage site is removed from a dimer A[C] and generates a 6-bp fragment of 1952.2 Da with the adjacent 4-bp fragment of 1318 Da.

Confirmatory information is generated from the reverse RNA transcript of the target region. Both the Tand the C-specific cleavage reaction generate mass shifts of —16 Da corresponding to an exchange of G vs. A in the affected fragment (Fig. 2).

In conclusion, a heterozygous sequence change can generate up to five discriminatory observations in a mass spectrum by adding or removing a cleavage site, as well as shift the mass of single products by the mass difference of an exchanged nucleotide. Up to 10 observations might be the result of a homozygous sequence change, because not only additional but also missing signals can be utilized for SNP identification.

Mono-, di-, and trimer nucleotides are usually noninformative. They are excluded from the analysis because of coinciding fragments, and their detection is also diminished because of analyte carrier matrix signals within the low mass range.

The combined observations of all cleavage reactions allow for the unambiguous detection, identification, and localization of almost all sequence changes. The inherent redundancy of information from all cleavage reactions substantiates the reliability of the results. Additional supportive information is obtained when signals are correlated across a multitude of samples.

A simulation of arbitrary 500-bp amplicons in the human genome showed that about 99% of all theoretical sequence changes can be detected and characterized.1-12-1

A homogeneous 384-assay format enables automated processing with liquid handling devices. Nanoliter dispensing onto matrix-coded chip arrays are utilized for automated, reproducible MALDI-TOF measurements. TOF instruments acquire data with turnaround times of 2 sec per sample at a standard 20-Hz laser repetition rate as opposed to hours of analyte separation in conventional sequencing gel electrophoresis. A single MALDI-TOF mass spectrometer can thus scan 2.5 million bp in every 24 hr.

Fig. 3 Pathogen identification by mass spectrometry. (A) Overlay of mass spectra of C-specific forward 16S rDNA fragments of Mycobacterium tuberculosis ATCC27294, Mycobacterium xenopi DSM43995, Mycobacterium paraffinicum DSM44181, and Mycobacterium gordonae DSM44160. Identifier peaks are marked with arrow. (B) Barcode of C-specific forward 16S rDNA discriminatory mass fragments.

Fig. 3 Pathogen identification by mass spectrometry. (A) Overlay of mass spectra of C-specific forward 16S rDNA fragments of Mycobacterium tuberculosis ATCC27294, Mycobacterium xenopi DSM43995, Mycobacterium paraffinicum DSM44181, and Mycobacterium gordonae DSM44160. Identifier peaks are marked with arrow. (B) Barcode of C-specific forward 16S rDNA discriminatory mass fragments.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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