DNA Sequencing by T.S. Work and R.H. Burdon (Eds.)

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By T.S. Work and R.H. Burdon (Eds.)

This quantity presents a accomplished description of the foundations and strategies utilized in DNA sequencing. Following a close advent the chapters are: DNA sequencing; Chain terminator sequencing; Primed synthesis equipment utilized to DNA fragments cloned into phage M13; DNA sequencing through the Maxam-Gilbert chemical technique; desktop equipment for DNA sequencers; Appendices together with contractions and precise phrases, cloning vectors, commercially to be had restrict endonucleases, and autoradiography.

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Coli to man), millions of individually packaged and replicated phage A recombinants can be stored in a single solution. AB h a s been extensively used for the replication of cloned DNA sequences from warm-blooded animals. Detailed reviews describing the varieties of plasmids and phages which are used for cloning and amplifying DNA sequences are found in Methods in Enzymology, (1979), Vol. 68. This whole volume is devoted to techniques used in recombinant DNA. Phage M13 and its derivatives This is described briefly since it is now the vehicle of choice for obtaining single-stranded recombinant DNA molecules for sequencing by the primed-synthesis method described in Chapter 4.

1. 2 DNA SEQUENCING 47 subsequently used to remove the primer and generate a unique 5'-terminus on the cDNA. However, as pointed out earlier, some restriction enzymes are strongly inhibited by the single stranded regions present in the template and cannot be used to cleave at the restriction site. An additional problem arises if a second cleavage site for the same enzyme is present within the sequence copied into the radioactive DNA. Two sets of fragments would be generated and a unique sequence would not be obtained.

0 unit) of the datum restriction endonuclease to each sample, mix, and incubate at 37°C for 30min. ) Stop the reaction by blowing into 10 p1 formamide-dye mix. - In addition, t o provide the reference pattern of oligonucleotides (the zero channel) a sample of the radioactive polynucleotide prepared in Step 3 is also digested with the restriction endonuclease. 0 unit) Incubate 37"C, 30 min Stop reaction by blowing into lop1 forrnamide-dye mixture. Step 5 : Gel electrophoresis (i) Preparation of the acrylamide gel This is conveniently done while the extension product is drying (step 3).

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