Alkylation of cysteine with acrylamide for protein sequence analysis.

Alkylation of cysteine in proteins with acrylamide under mildly alkaline conditions yields a thioether derivative, Cys-S-beta-propionamide (Cys-S-Pam), which is stable during automated Edman degradation. Its phenylthiohydantoin derivative, PTH-Cys-S-Pam, is easily separated from other PTH-amino acids by HPLC and is thus useful for cysteine identification during protein sequencing. PTH-Cys-S-Pam was first noticed during sequencing polypeptides blotted onto polyvinylidene difluoride membranes from polyacrylamide gels, in which cysteine had reacted with residual unpolymerized acrylamide. Cysteine in proteins is easily alkylated by reaction of proteins in aqueous solution with acrylamide. Methods are also presented for alkylation of cysteine in proteins adsorbed on fiberglass disks in the reaction cartridge of a protein sequencer. Finally, PTH-Cys-S-Pam was synthesized chemically. The synthetic compound is unstable in neutral solution, but can be stabilized by acidification. It has the same HPLC retention time as the product formed from cysteine when sequencing proteins alkylated with acrylamide.     

Direct protein microsequencing from Immobilon-P Transfer Membrane

Proteins separated by electrophoresis and electroblotted onto Immobilon-P Transfer Membrane can be sequenced directly in the gas-phase sequencer. Protein bands visualized by Coomassie Blue are placed in the sequencer cartridge without the addition of polybrene. Preconditioning sequencer cycles are eliminated, reducing reagent use and instrument operating time. The average initial yield for protein spotted or blotted onto the polyvinylidene-based membrane was determined to be 70 to 80% using 125I-labeled beta-lactoglobulin. Preliminary data indicate that proteins hydrolyzed in situ on Immobilon-P can further be characterized by amino acid compositional analysis.     

Footprinting with an automated capillary DNA sequencer

Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.     

Specific-primer-directed DNA sequencing using automated fluorescence detection.

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.     

ABI sequencing analysis

The ABI Sequencing Analysis application is designed specifically for the analysis of data produced by the ABI DNA Sequencer. The ABI sequencer is a laser-based instrument that utilizes fluorescent labels to analyze the products of a sequencing reaction as they migrate through a gel. After the data are collected from a sequencing run, the Analysis program identifies and tracks the sample lanes of the gel and subsequently normalizes and integrates the raw data into a chromatogram of the final sequence. For the use, there are basically two types of files that can be manipulated to potentially improve the analysis results. The Gel File consists of a computer generated image of the sequencing gel with the fluorescent DNA banding patterns. This image allows the user to view and edit the tracking lines generated and used by Analysis to collect data points for each sample. Individual Sample Files are stored for each of the samples analyzed and include the chromatogram, raw data, and annotations and information regarding the sample and sequence run. Generally, the products of a sequencing reaction are easily resolved and the Analysis software interprets the correct nucleotide sequence. Ambiguous base calls tend to occur near the end of the sequence and may be either edited or deleted by the user before exporting the data for further comparisons or alignments. Occasionally the tracking lines within the gel image may need to be adjusted or moved. The sample data are then reextracted from the Gel File and analyzed again. This review explains the general operation of Analysis in terms of viewing and editing a chromatogram, retracking the lanes of a Gel File, and analyzing the final sample data. The three versions 1.2.1, 2.1.2, and 3.3 are discussed.     

Typing single-nucleotide polymorphisms using a gel-based sequencer

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot™ kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotype (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.     

Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot trade mark kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotyper (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.     

Automated four-color DNA sequencing using primers assembled by hexamer ligation

A procedure based on the assembly of sequencing primers by hexamer ligation and then using them in automated DNA sequencing is described. This method is based on a four-color fluorescent terminator chemistry. Sequencing ladders were analyzed using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The best results were obtained for primers assembled by ligation of four to ten hexamers. The accuracy of the method was estimated to be 99.5% up to 400 nt of the read sequence, and somewhat lower at 400–600 nt.     

Loader Lite: a new software tool for the ABI PRISM 3700 DNA sequencer

Here we describe the development of a novel software tool entitled Loader Lite that generates plate records or sample sheetsfor the ABI PRISMs 3700 DNA sequencer. The major advantage of this program is that it enables the ongoing operation of sequencing instruments without reference to external network(s). The autonomous operation of sequencing instruments is critical if sample throughput is to be maintained during periods of network outage. Loader Lite employs a deliberate strategy of inputting anonymous tray barcodes at run time. After sequencing, the barcodes are reconciled with relevant project details by reference to a database. This software takes advantage of barcode scanning technology by creating plate records directly on the local computer, serving an individual sequencer, immediately before importing and linking. This real-time synthesis of the plate records at the point of loading all but eliminates loading errors. Loader Lite is user-friendly, fully configurable, and permits the running of partial or full 384-well sample trays, using any standard combinations of run modules, dye sets, mobility files, analysis modules, etc. The 96-well format is not supported; however, this capability will appear in subsequent versions that are currently under development. This application is designed as an added value, adjunct program to the regular ABI PRISM 3700 Data Collection software. We have successfully used Loader Lite over the past six months to load approximately 7 million sequencing reactions and believe its utility and functionality will prove to be attractive to the wider sequencing community.     

Two-step cycle sequencing improves base ambiguities and signal dropouts in DNA sequencing reactions using energy-transfer-based fluorescent Dye terminators

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods plays an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While dideoxy-terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity from that point on in the sequence. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrate that combining the annealing step with the extension step at one temperature (60°C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used. This modification significantly increases the number of accurately read bases in DNA sequences.     

safum: statistical analysis of SSCP fingerprints using PCA projections,dendrograms and diversity estimators

The program safum provides a smart interface to import, visualize and compare fingerprinting profiles, especially on capillary electrophoresis single strand conformation polymorphism data, in conjunction with basic statistical analysis tools. It includes principal component analysis, two‐ or three‐dimensional representations, dendrograms based on Euclidean distance, and easily exportable files for subsequent applications. safum is useful for the analysis of spatial or temporal sequences of microbial community fingerprints obtained with an ABI prism sequencer.     

On-sequencer pyridylethylation of cysteine residues after protection of amino groups by reaction with phenylisothiocyanate

Cysteine residues in polypeptides are not easily identified during automated N-terminal sequence analysis. Reaction of cysteine side chains with 4-vinylpyridine and identification as the pyridylethylated phenylthiohydantion derivative (PE-PTH-Cys) were proposed. However, after this reaction a desalting step is necessary. If limited sample amounts do not allow this desalting step, on-sequencer pyridylethylation is an alternative, although preview of the consecutive amino acid is usually observed in this case. We describe an on-sequencer procedure that avoids such preview formation by derivatizing the peptide with phenylisothiocyanate (PITC) prior to reaction with 4-vinylpyridine. The pyridylethylation is performed in the cartridge of the sequencer after immobilization of the protein or peptide on a polybrene-coated glass fiber filter and thiocarbamylation with PITC. Preview caused by N-alkylation is not observed and PE-PTH-Cys is detected in much higher yields than usual. The procedure reported here is significantly shortened, optimized to reduce side products, and avoids losses during sample handling. It can easily be adapted to any automated version of the sequencers.     

Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with edman degradation

Use of a C-terminal sequencer with modified solvents, reagent concentrations, chromatographic parameters, temperatures, and reaction cartridge geometry yields four sets of improvements in chemical degradations. They are increased sensitivity, longer runs, passage of Pro residues, and practical combination with N-terminal degradation. Over 200 proteins and protein fragments with sizes between 20 and 600 residues were analyzed. C-terminal sequences could be interpreted for more than 10 residues at high picomole sample levels, while the 10-pmol level gave 4-5 residues. The average initial yield was 15% but up to 30% could be achieved. The improved performance allowed combination of C- and N-terminal degradations from the same sample application. After initial Edman degradation, the sample is moved to the C-terminal instrument for continued sequencing. Proteins available in limited amount are thereby efficiently analyzed. Lys, modified from the N-terminal degradation, may be detected as the alkylated thiohydantoin-phenylthiocarbamyl-Lys derivative in the C-terminal degradation. Notably, C-terminal sequence analysis could be proceeded through Pro residues which unexpectedly were no absolute hindrance. The improved technique provides characterization of truncation patterns and microheterogeneities in proteins down to the 10-pmol level and is a useful approach for analysis of N-terminally blocked polypeptides.     

细胞因子人MK基因的克隆及在大肠杆菌中的高效表达(简报)

细胞因子Midkine(简称MK)是新发现的一类肝素结合因子家族中的一员。1988年,Kadamatsu等利用差异杂交法在经维甲酸诱导分化的小鼠畸胎瘤细胞株HM-1中首先克隆到小鼠MK基因。人MK基因则最早是从λgt10人胚肾(20-24周)cDNA库和EMBL-3人胎盘基因组库获得。成熟     

细胞因子人MK基因的克隆及在大肠杆菌中的高效表达（简报）

For cloning the cytokine human Midkine (MK) gene, we designed by PCgene program and synthesized a pair of PCR specific primers according to the reported human MK cDNA sequence. Total cellular RNA was extracted from a human hepatoblastoma cell line HepG2, and then the target DNA fragment was obtained by RT-PCR and subcloned into plasmid pUC118. Checked with radioisotope sequencing and ABI 377A sequencer, the nucleotide sequence of the cloned MK cDNA was identical with the reported one. A prokaryotic expression vector, named pBV220, was used to express the MK protein efficiently in E. coli strain TG1 and a predicted band of 16.5 kD in Mr by 15% SDS-PAGE was found. The expressed recombinant protein was found in insoluble aggregated form and accounted for about 31.21% of the total cellular proteins. The first 15 N-terminal amino acid sequence analysis of this protein by Edman degradation method showed that it was accordant with that predicted from the cDNA sequence. The activity of neurite outgrowth-promoting of the MK crude samples was tested with brain cells isolated from 18-day embryos of SD rat.     

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.     

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.     

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.