Direct sequencing of long polymerase chain reaction fragments

Direct sequencing of polymerase chain reaction (PCR)-generated templates is a commonly used technique in molecular biology laboratories. We describe an improved method for direct sequencing of PCR fragments longer than 20 kb obtained with a commercial mixture ofTaq andPwo DNA polymerases. The sequencing protocol was optimized for an automated infrared DNA sequencer, consistently yielding long reads (500–600 bases).     

A high-throughput, low-cost method for the preparation of "sequencing-ready" phage DNA template

We have developed a simple and efficient protocol for the isolation of good-quality recombinant phage DNA useful for all downstream processing, including automated sequencing. The overnight-grown phage particles were effectively precipitated (without any contaminating Escherichia coli DNA and other culture media components) by adjusting the pH of the culture medium to 5.2 with sodium acetate, followed by addition of ethanol to 25%. The phage DNA was selectively precipitated with ethanol in the presence of guanidinium thiocyanate under alkaline pH, resulting in uniform quality and quantity of phage DNA. The quality of the phage DNA preparation was demonstrated by DNA sequencing that provided an average read length of >700 bases (PHRED20 quality). This protocol for plating, picking, growing, and subsequent DNA purification of individual phage clones can be completely automated using any standard robotic platform. This protocol does not require any commercial kits and can be completed within 2 h.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.     

A non-radioactive automated method for DNA sequence determination

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.     

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.     

Direct visualization of site-specific and strand-specific DNA methylation patterns in automated DNA sequencing data.

We report here a simple method of directly visualizing in automated DNA sequencing chromatograms DNA methylations of different types including cytosine methylations in Hpa II and dcm sites as well as adenine methylations in dam sites. This is made possible by the observation that the extent of incorporation of fluorescently labeled dideoxynucleotides is influenced by the methylated bases in template DNA. This simple approach involves routine automated DNA sequencing without any prior treatment of DNA specific for detecting DNA methylation.     

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries.

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.     

Modifications of guanine bases during oligonucleotide synthesis.

Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions. Methods for the detection of two types of guanine modifications are described. The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing. The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA. Though the chemical structures of the two modifications are not known, they may be related. Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry. The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W. (1980) Nucleic Acids Res. 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases. We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.     

Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography

This work describes the application of the fully automated high-performance liquid chromatographic system to the analysis of PCR-amplified products. Efficient separations of both DNA restriction fragments and PCR products were performed using an anion-exchange DEAE-NPR column, packed with 2.5-μm nonporous particles. The automated HPLC method was employed for the separation, quantitation, and purification of PCR products in less than 10 min in a single step.     

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.     

A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer

A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5'-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication.     

Analysis and purification of synthetic large oligodeoxyribonucleotides by HPLC on RPC-5 like resin

Large oligodeoxyribonucleotides (20-160 bases), synthesized by the phosphoramidite method, have been analyzed and purified by HPLC on a RPC-5 like resin (Neosorb LC). Linear gradient of NaClO4 solution containing 10 mM NaOH and 0.1 mM EDTA was carried out for the elution. Large oligodeoxyribonucleotides bearing 4,4'-dimethoxytrytyl (DMT) group were separated very well from the shorter failed by-products. After removal of the DMT group, the products were analyzed and purified by repeating HPLC on Neosorb LC. This HPLC system gave well resolution of the desired oligodeoxyribonucleotide (over 50 bases) from the base modified by-products with the same chain length. The chromatogram showed the presence of large amount of by-products in addition to the desired product when methylphosphoramidite method was employed for the DNA synthesis.     

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.     

Manganese citrate improves base-calling accuracy in DNA sequencing reactions using rhodamine-based fluorescent dye-terminators.

While dideoxy-terminators labeled with rhodamine-based fluorescent dyes provide the most versatile method of automated DNA sequencing, variation in peak heights reduces base-calling accuracy. We describe a simple approach that uses additions of a manganese salt and the metal buffer sodium citrate (MnCit) to overcome this limitation. This modification reduces peak height variability >2-fold and significantly increases the number of accurately read bases in DNA sequences.     

Silanized nucleic acids: a general platform for DNA immobilization

We have developed a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products on unmodified glass surfaces. By covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solutions we have generated a new class of modified nucleic acids, namely silanized nucleic acids. Such silanized molecules can be immobilized instantly onto glass surfaces after manual or automated deposition. This method provides a simple and rapid, yet very efficient, solution to the immobilization of prefabricated oligonucleotides and DNA for chip production.     

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

Background  

Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

Automated Sanger DNA sequencing with one label in less than four lanes on gel

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.     

Biological consequences of free radical-damaged DNA bases

The principal oxidized cytosine bases, uracil glycol, 5-hydroxycytosine, and 5-hydroxyuracil, are readily bypassed, miscode, and are thus important premutagenic lesions. Similarly the principal oxidation product of guanine, 8-oxoguanine, miscodes with A and is a premutagenic lesion. Most of the thymine and adenine products that retain their ring structure primarily pair with their cognate bases and are not potent premutagenic lesions. Although thymine glycol pairs with its cognate base and is not mutagenic it significantly distorts the DNA molecule and is a lethal lesion. Ring fragmentation, ring contraction, and ring open products of both pyrimidines and purines block DNA polymerases and are potentially lethal lesions. Although these breakdown products have the potential to mispair during translesion synthesis, the mutational spectra of prokaryotic mutants defective in the pyrimidine-specific and/or purine-specific DNA glycosylases do not reflect that expected of the breakdown products. Taken together, the data suggest that the principal biological consequences of endogenously produced and unrepaired free radical-damaged DNA bases are mutations.