DNA sequence protocols

Griffin, A.M., Griffin, A.G. (Eds.) (1994) DNA-Sequencing Protocols, in Methods in Molecular Biology Vol. 23. Humana Press, Totowa, NJ. 

Sears, L., Moran, L, Kissinger, C., Creasey, T O Keefe, P., Roskey, M., Sutherland, E., Slatko, B. (1992) CircumVent Thermal Cycle Sequencing and Alternative Manual and Automated DNA Sequencing Protocols Using the Highly Thermostable VentR (exo-) DNA Polymerase, BioTech-niques 13, 626-633. 

DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill,... 

DNA Sequencing Protocols, edited hyHugh G. Griffin ondAnnetteM. Griffin, 1993... 

DNA Sequencing Protocols, Second Edition, edited by Colin... 

Design qualification (DQ) is the process used to determine a system that will function within the intended purpose. It can be compared to a user s requirements document for a piece of software. For example, if a CE is being purchased to run DNA sequencing samples, then the system purchased will need to include a fluorescence detector. The main vendors for CEs have similar options for their CE instruments reducing the utility of DQ protocols. Their main utility is to define the specific equipment needed for the purchase order. This only needs to be performed before the system is purchased initially. If desired, this can also be done when additional features need to be purchased (i.e., new detectors, etc.). 

Most gene therapy protocols currently under way involve the replacement of a missing gene product in a cell (e.g., the replacement of clotting fitctor Vin in a hemophilia A patient). Recombinant DNA techniques (see Section I, Chapter 6 Recombinant DNA and Gene Cloning) are used to insert a normal DNA sequence into a mJw, wUdi dm carries the DNA into the patient s cells, where it suppUes a template fiir the normal gme prodnct (KgU-6-2). 

Frommer, M., McDonald, L. E., Millar, D. S., et al. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U. S. A. 89,1827-1831. 

Nowadays ChIP is a standard technique to determine whether a protein of interest (or a modification of the protein) is present at a specific DNA sequence in vivo. Figure 7.1 shows the basic steps of the standard ChIP protocol. It consists of... 

Since protocols for electrophoresis used in DNA sequence analysis are given, for example, by Sambrock et al., these specialized methods are not outlined in this chapter. 

In a study by Sisk et al. (1994), male B6C3Fj lad transgenic mice were exposed by inhalation to 0, 62.5, 625 or 1250 ppm [0, 138, 1380 or 2760 mg/m ] butadiene for four weeks (6 h per day, five days per week). Animals were killed 14 days after the last exposure and lad mutants were recovered from the DNA according to established protocols. A 2.5- and 3-fold increase in the lad mutant frequency was observed in the bone marrow of mice exposed to 625 or 1250 ppm butadiene, respectively, compared with air-exposed control mice. DNA sequence analysis of lad mutants recovered from the bone marrow of mice exposed to 625 ppm butadiene demonstrated that there was a shift in the spectrum of base substitution mutations at A T base pairs in butadiene-exposed mice (6/26, 23%), compared to air control mice (2/45, 4%). Recio and Meyer (1995) examined the lad mutational spectrum in the bone marrow of mice exposed to 1250 ppm butadiene in the above study. DNA sequence analysis of lad mutants revealed an increase in mutations at A T base pairs (9/49, 20%) similar to that observed by Sisk et al. (1994). 

DNA sequences can be determined and DNA polymers synthesized with simple, automated protocols involving chemical and enzymatic methods. 

Comparison of a normal cloned gene with an uncloned mutant form of the gene PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laboriously cloning the altered DNA. 

Kit for DNA sequencing Available with protocol from commercial suppliers. 

Both Taq and Pfu polymerases are suitable for this protocol. In general, Taq gives more consistent and greater yields but can also introduce undetected mutations. Pfu is less error prone, but we find it to be more sensitive to specific DNA sequences and conditions. 

A typical PCR reaction set up protocol is shown in Table 6.3. The first step in PCR involves the selection of oligonucleotide primer sequences that are optimal for the amplification of a particular target DNA sequence. In addi-... 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

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