Recent advances in DNA sequencing methods - general principles of sample preparation

DNA sequencing has revolutionized biomedicine, and progress in the field has been unrelenting since it was invented over 30 years ago. The complete DNA sequence of the human genome was obtained as the culmination of a decade of work by a large number of scientists. Less than ten years later, so-called ‘next-generation’ instruments now make it possible for a single lab to produce the same amount of data in a week. But while the instruments are increasingly automated, upstream sample processing remains a challenge. Here I review the current state of the art in preparing genomic and RNA samples for high throughput sequencing.     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Affinity chromatography—dependent selection （ACDS） of genomic DNA fragments bound specifically to bacterial synthesized Myc／Myn proteins

This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among genomic DNA.cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells.DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vector respectively.Fusion GST-Myc and GST-Myn synthesized in E.coli hosts showed affinity to CACGTG E-box DNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR.A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA.At least two genomic DNA fragments obtained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone.Significance of the work and of the technique itself as well asidentification of the DNAs are discussed.     

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.     

An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR

A simple method for removing contaminating genomic DNA from an RNA preparation is presented. The method involves digestion of the RNA with RNase-free DNase I at room temperature followed by inactivation of the enzyme at 65°C in presence of EDTA. This method produces an RNA sample that is negative for genomic DNA by PCR.     

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.     

Molecular and chromosomal organization of two repetitive DNA sequences with intercalary locations in sugar beet and other Beta species

 In a search for repetitive DNA sequences in the sugar beet genome, two sequences with repeat unit lengths of 143 and 434 bp were isolated and characterized. The pSV family showed an unusual conservation of restriction sites reflecting homogenization of the analyzed repeats. Members of the family are organized as tandem repeats as revealed by PCR and sequencing of dimeric units. The pSV satellite occurs in large intercalary arrays which are present on all chromosome arms of sugar beet. The pSV sequence family is present in different abundance in the sections Beta, Corollinae and Nanae but is not detectable by Southern hybridization in the section Procumbentes. The pDRV family is characterized by an interspersed genomic organization. The sequence is detectable in all sections of the genus and is amplified in species of the section Beta but was also detected, although at lower abundance, in the remaining three sections. Fluorescent in situ hybridization has shown that the pDRV sequence family is dispersed over all chromosomes of the sugar beet complement with some regions of clustering and centromeric depletion. Received: 18 March 1998 / Accepted: 31 March 1998     

Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.     

Determination of amphetamine and methamphetamine enantiomers by chiral derivatization and gas chromatography–mass spectrometry as a test case for an automated sample preparation system

An automated sample preparation system has been applied to the chiral analysis of amphetamine and methamphetamine using derivatization with trifluoracetyl-L -prolyl chloride (L -TPC) and subsequent separation on a gas chromatography–mass spectrometry (GC-MS) system. Tasks automated were the dilution of standards and the off-line preparation of the diastereoisomer derivatives. Chromatographic performance, sensitivity, and reproducibility of the automated procedure were compared to the equivalent values obtained with two existing assays methods which employ manual derivatiation, either on-column or off-line. Chromatographic performance was unaffected by the derivatization procedure and sensitivity was better for both automated and manual off-line derivatization. Qualitative reproducibility as based on enantiomeric composition was equivalent for all three approaches, while quantitative reproducibility as based on peak areas was best for the automated procedure. Considering the fact that the diastereoisomer derivatives are unstable over time, automated sample preparation with “just-in-time” derivatization can increase the overall precision of the analytical method. The procedures described here are general enough in nature that they could be applied to other chiral or even achiral analytes. © 1994 Wiley-Liss, Inc.     

Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes.

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time-consuming procedures, and with them, we cannot "walk" chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end-specific "contextual" sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination with cloning of the inverse PCR-generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in "walking" chromosomes.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system

We propose a novel method to prepare a DNA–protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni²⁺. Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA–protein conjugate was formed and immobilized in the presence of Ni²⁺ on the microplate. We then adopted alkaline phosphatase (AP) as a model protein, and application of the DNA–AP conjugate was demonstrated in a thrombin aptamer-based detection system with a detection limit of approximately 10 nM.     

An Assessment of Oxidative Damage to Proteins, Lipids, and DNA in Brain from Patients with Alzheimer's Disease

Abstract: Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.