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Generation of Random Fragments by Sonication

Except for all but the shortest of sequencing projects, the resolution limits of polyacrylamide gels (being somewhat less than 1 kb) prevents determining the entire sequence in one run. There are several ways to overcome this by subcloning or by primer walking. Each has its advantages and disadv ...

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M13 Phage Growth and DNA Purification Using 96 Well Microtiter Trays

The growth and purification of M13 DNA from small volume (1.5mL) cultures is a rapid and easily performed procedure (1). The samples can be processed in microcentrifuges in disposable polypropylene tubes and yield sufficient, pure single-stranded DNA (4 μM) for five or more sequencing exper ...

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The Use of Robotic Workstations in DNA Sequencing

With the advent of genome sequencing, a much greater impetus has taken effect in expanding the capabilities of DNA sequencing. This is necessary since present limitations make such projects very long term and very expensive. Although this drive shall inevitably bring about a revolution in D ...

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M13 Phage Growth and Single-Strand DNA Preparation

Ml3 phages do not lyse their host, but are released from infected cells as the cells continue to grow and divide. Cells infected with Ml3 have a longer replication cycle than uninfected cells, and as the infection proceeds, areas of slower-growing cells can be visualized as turbid plaques on lawns of E. co ...

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Sequencing Using the Du Pont Genesis™ 2000 DNA Analysis System

The Du Pont Genesis™ 2000 DNA Analysis System consists of an instrument, reagents capable of labeling DNA with novel fluorescent dideoxynucleotides (1), and a Macintosh® II computer to operate the instrument and perform all subsequent data analysis The instrument houses a vertical elec ...

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Sequencing Reactions for ALF (EMBL) Automated DNA Sequencer

Automated fluorescent DNA sequencing (1–3) has turned into a subject of great interest during the last few years, and has successfully been applied to large-scale sequencing projects, like sequencing of the human HPRT gene locus (4). Most actual genome sequencing efforts, such as sequencing ...

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Transfection of E. coli with M13 DNA

In recent years several techniques have been described for the introduction of DNA molecules into strains of E. coli by transformation or transfection. These are based on the findings of Mandel and Higa (1) who demonstrated that incubation of cells with naked bacteriophage λ DNA in cold calcium c ...

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Sequencing Reactions for the Applied Biosystems 373AAutomated DNA Sequencer

Efficient completion of large DNA sequencing projects has been greatly facilitated by the development of fluorescence-based dideoxynucleotide sequencing chemistries and instruments for real-time detection of fluorescence-labeled DNA fragments during gel electro ...

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Linear Amplification Sequencing with Dye Terminators

Present day sequencing technology is mainly based on the dideoxy method introduced by Sanger in 1977 (1;see ). DNA sequencing is a complex process requiring many different complicated steps like subcloning, template preparation, sequencing reactions, gel electrophoresis, readi ...

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DNA Sequencing by Chemical Degradation Using One, Two, and Four Different Fluorophores

There are several chemical methods for sequencing DNA. All of them are based on gel separation of cleaned fragments. However, they use different chemistries to generate these fragments.

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DNA Sequencing the Chemical Method

The chemical method of sequencing DNA (1) has some advantages and some disadvantages compared with the enzymatic method (2). The major disadvantage is that it takes more time to produce the same amount of sequence. This is so for two main reasons. First, the DNA has to be end-labeled and then reisolated pr ...

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Terminal Labeling of DNA for Maxam and Gilbert Sequencing

There are two basic enzymatic activities that are used to end-label DNA with radioactive phosphate (32P). The enzyme T4-polynucleotide kinase will use the substrate ATP to add a phosphate group (the gamma-phosphate of the ATP molecule) preferentially to the 5′ ends of the molecule. The enzyme DNA ...

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Reverse Sequencing of M13 Cloned DNA

When shotgun sequencing projects are almost complete, continued random sequencing becomes increasingly less fruitful. The sequence may have regions of data that have only been determined on one strand (and therefore potentially error prone) or there may be gaps in the sequence. In both of th ...

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DNA Sequencing by Chemiluminescent Detection

The development of fluorescent, calorimetric, and more recently chemiluminescent detection systems have opened the way to replace radioactive detection for DNA sequencing (1–3). Although the bulk of DNA sequencing is still carried out using radioactive detection, the availabil ...

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Cloning into M13

The bacteriophage M13 has been developed into a cloning vector system for obtaining single-stranded DNA template required for the dideoxy chain termination method of sequencing DNA (1,2). General aspects of bacteriophage M13 as a cloning vector system are reviewed in Chapter 2, and the pre ...

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Multipllex DNA Sequencing

Various approaches have been proposed to speed up the process of DNA sequencing in order to facilitate sequencing of large genomes from bacteria (~4 Mbp), yeast (~13 Mbp), human (~3000 Mbp), and other organisms (1-3). The multiplex DNA sequencing strategy (4) presents one of these approaches. It is com ...

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Direct Blotting Electrophoresis

The analysis of membrane-immobilized macromolecules such as DNA/RNA and proteins presents a common analytical method with widespread applications. The macromolecules are either spotted directly onto a membrane or, if they are a complex mixture, they are separated by electrophor ...

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Cycle Sequencing

Cycle sequencing (1,2) is a simple, yet powerful tool for conveniently sequencing double-stranded DNA (dsDNA). As in other dideoxy sequencing methods (3–5) dsDNA is denatured, a primer is annealed, then a complementary oligonucleotide is synthesized by a DNA poIymerase until extension is ...

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Solid Phase PCR Sequencing of Biotinylated Products

The dideoxy sequencing method (1) has been universally employed and utilized for a wealth of sequencing projects such as small (2) and larger viral genomes (3), and requires purified M 13 or plasmid DNA templates. Numerous improvements have been made to obtain more DNA sequence from a single clone, i ...

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Sequencing Double-Stranded Linear DNA with Sequenase and [α-35S] d.ATP

The Polymerase Chain Reaction (PCR) products are doublestranded linear DNA molecules. Although hybridization may provide some information on the amplified products, clearcut identification of nucleic acids is best achieved by sequencing. When PCR fragments are heterogeneo ...

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