In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.     

Direct sequencing of PCR-amplified DNA

This article describes the direct sequencing of PCR-amplified DNA, a technique that bypasses the problem of replication errors sometimes associated with other PCR procedures. The direct sequencing procedure produces an “average sequence” of all the copies of the target. Any miscopied molecule usually represents only a small proportion of the total. The technique described here is based on the “traditional” ddNTP sequencing method of Sanger et al.     

A modified TnphoA useful for single-stranded DNA sequencing.

The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.     

The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction

A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.     

Production of single-stranded DNA for sequencing: an alternative approach.

We describe a simple procedure for the direct sequencing of single-stranded, PCR-amplified, target regions of human genomic DNA. At variance with previously reported procedures, purification of the desired double-stranded DNA was introduced. This additional step allowed the single-stranded amplification and sequencing of the target gene. This step is required for direct sequencing of some amplified regions of human genomic DNA. However, no individual technique seems suitable to generate and sequence all single-stranded DNA.     

Direct sequencing of unpurified PCR-amplified DNA by semi-exponential cycle sequencing (SECS)

A simple technique for direct sequencing of PCR-amplified templates without purification of the PCR reaction product is presented. This method does not require an additional synthesis step after template amplification, and can generate sequence information form as little as 0.1 fmol of unpurified template.     

Efficient preparation of single-stranded DNA for in vitro selection

In vitro selection of aptamers requires the reliable enzymatic preparation of large amounts of (+) single-stranded DNA molecules. This can be achieved by selective enzymatic digest of 5′-phosphorylated (?) strands from PCR products, a method already widely used in sequencing of PCR products. Here we present an adaptation of this method to prepare large pools of single-stranded DNA molecules for in vitro selection.     

A method for sequencing single-stranded cloned DNA in both directions

A DNA sequencing method has been developed whereby DNA that has been cloned in a single-stranded bacteriophage vector can be sequenced from both ends. The method involves first making a minus-strand sense template from a single-stranded insert in the vector MJ3mp2 using a flanking primer, and then sequencing the synthesized template using the dideoxynucleotide termination method (Sangeret al., 1977, 1980) with a second primer. Special conditions are described under which the first primer is easily removed after making the templat% and sequencing in the opposite direction can be done in the normal way (Sangeret al., 1980) without separating the double strands. This method renders it possible to read up to twice the amount of sequence data from a long insert and also to check short inserts by producing complementary sequence patterns.     

Use of a novel agarose gel-digesting enzyme for easy and rapid purification of PCR-amplified DNA for sequencing.

The use of a novel gel-digesting enzyme preparation provides an easy, rapid and convenient method to quantitatively recover PCR-amplified DNA from low melting point agarose gels. The PCR products purified using this method were readily sequenced and yielded good and unambiguous sequence data.     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.     

Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization

This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.     

Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation

Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data.     

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.     

Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction

A method to rapidly generate single stranded DNA for dideoxy sequencing following the polymerase chain reaction is described. By incorporating biotin in one of the amplification primers, we are able to physically separate the two DNA strands produced in the polymerase chain reaction. After amplification, the mixture is passed through a column containing streptavidin agarose. The strand produced by the biotinylated primer is bound in this matrix. The unbiotinylated strand is eluted with 0.2 N NaOH and sequenced by the dideoxy method. This method was utilized to sequence mitochondrial DNA from crude genomic DNA and to determine the sequences of four clones containing human mitochondrial DNA as a test of its accuracy. The use of biotin-facilitated separation permitted us to amplify and sequence DNA samples in a single day.