Genome Analysis By Mass Spectrometry History And Stateoftheart

Analysis of nucleic acids by mass spectrometry has mainly been accomplished by two common soft ionization techniques—ElectroSpray Ionization (ESI) and Matrix-

Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS).

The use of MALDI coupled with time-of-flight mass spectrometry (MALDI-TOF MS) has become one of the leading technologies for high-throughput analysis and high-fidelity measurement of nucleic acid sequence variations. Unambiguous detection of known polymorphic sequences has been demonstrated, even in various for-mats.[3-5]

Early attempts to apply MALDI-TOF to de novo sequencing as an alternative to separation and detection of Sanger sequencing ladders[6] were hindered by physical analyte fragmentation, limited mass resolution, and mass accuracy in the high mass range. Despite several promising biochemical strategies, which generate truncated DNA sequence fragments of a sequencing primer analogous to dideoxy sequencing, routine-read lengths exceeding 100 bp have never been achieved.[7-9]

In addition to the primer extension-based Sanger sequencing approach, several chemical and enzymatic DNA fragmentation approaches have been proposed to generate short-based specifically cleaved MALDI-TOF analytes.

A chemical cleavage approach utilized P3'-N5'-phosphoamidate-containing DNA replacing dCTP or dTTP by their analog P-N modified nucleoside triphos-pates. Acidic reaction conditions induce base-specific cleavage. However, the required acidic conditions produce unwanted depurination by-products and base loss of adenine and guanine.[10]

A uracil-DNA-glycosylase (UDG)-treatment approach uses strand-separated polymerase chain reaction (PCR) products to generate T-specific abasic sites. Subsequent alkaline and heat treatment induces base-specific DNA cleavage at each T-specific positions.[11] DNA regions of interest require incorporation of dUTP instead of dTTP during PCR. Strand separation is performed by solidphase separation on streptavidin-coated magnetic beads, which complicates the automatic handling of the assay.

Homogeneous assay formats requiring only subsequent addition of reagents are preferred. Post-PCR in vitro transcription systems combined with base-specific cleavage of the RNA transcripts overcome the issues encountered with classical DNA amplification and primer extension reactions.

The current state-of-the-art differential sequence analysis concept uses a Maxim-Gilbert-like approach: base-specific cleavage of nucleic acid amplification products. PCR amplification of the locus of interest is followed by in vitro transcription and base-specific cleavage.[12] This novel comparative resequencing scheme combines a homogenous in vitro transcription/RNAse system with MALDI-TOF analysis of molecular fragment masses—an intrinsic molecule property. No labeling is required. PCR products of up to 1 kb are subjected to in vitro transcription. Subsequent cleavage of the in vitro tran scripts by ribonucleases (e.g., RNase A) generates base-specifically cleaved RNA fragments. Sequence-specific mass signal pattern within a mass range of 1000-9000 Da—equivalent to 3-30 nucleotides—are obtained. The result is a characteristic pattern of RNA fragment masses indicative of the original reference sequence.

RNase T1 (a guanine-specific endonuclease) cleaves in vitro transcripts base—specifically at every G-position.[13] An available alternative is RNase A, which cuts specifically at the 3'-end of the pyrimidine residues C and U.

RNA analytes are more stable and less prone to depurination than DNA during the desorption/ionization process in MALDI-TOF MS because of a balancing effect

Target Maldi

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.

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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