DNA sequence analysis

Large DNA 5555555 Double-stranded (ds) DNA fragments resolve on polyacrylamide get 

Each 5 -labeled single-stranded DNA is then sequenced. The method is based on two principles  

In a typical experiment a portion of the 5 -labeled single-stranded DNA is reacted with dimethylsulfate under conditions where many but not all of the purine bases react. This reagent alkylates the N7 position 

An analogous series of reactions is used to produce depyrimidinated DNA fragments. Hydrazine is used in these reactions, since both cytosine and thymine react with hydrazine. The bases are cleaved to yield urea and a pyrazole ring. The deoxyribose moiety is left as a hydrazone. Piperidine, which reacts with the hydrazone, is used to cleave the nucleotide chain. Cytosines react specifically with hydrazine in 5 mol/ L NaCl, but no specific reaction exists for thymines. Consequently, one aliquot yields labeled oligonu-cleotides 3 -terminated at cytosines, whereas a second aliquot contains nucleotides cleaved in the absence of NaCI at both cytosine and thymine residues. 

Sequences of more than 350 bases can be obtained from some gels. The sequence of the complementary DNA strand is often determined as a check on accuracy of the first analysis. The primary structure of thousands of deoxyribonucleotides can be deduced by the analysis of 

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Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

Schoffl, F., Raschke, E. Nagao, R.T. (1984). The DNA sequence analysis of soybean heat-shock genes and identification of possible regulatory promoter elements. EMBO Journal, 3, 2491-7. 

The calculated value for the Hisg-tagged protein is 28,492 Da. N-terminal amino acid sequence analysis (Procise) was carried out on purified recombinant resilin. The following sequence (at 120 pmol yield) was obtained for the first 12 amino acid residues MHHHHHHPEPPV, as expected from DNA sequence analysis. 

The DNA sequence analysis of specific genes has allowed the recognition of a number of sequences important in gene transcription. From the large number of bacterial genes smdied it is possible to construct consensus models of transcription initiation and termination signals. 

Holland MM, Fisher D, Mitchell LG, Rodriquez WC, Canik JJ, Merril CR and Weedn VW (1993) Mitochondrial DNA sequence analysis of human skeletal remains identification of remains from the Vietnam War. J Forensic Sci 38 542-553. 

Holland MM, Fisher DL, Roby RK, Ruderman J, Bryson C and Weedn VW (1995) Mitochondrial DNA sequence analysis of human remains. Crime Lab Digest 22 109-115. 

Sedlmeier, R. and Altenbuchner, J., Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans, Mol Gen Genet, 236 (1), 76-85, 1992. 

Klein CA, Schmidt-Kittler O, Schardt JA, et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc. Natl. Acad. Sci. USA 1999 96 4494-4499. 

Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hood, L.E. (1985) The synthesis of oligonucleotides containing an aliphatic amino group at the 5 terminus Synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 13, 2399-2412. 

P.A. Caviani, D. Solas, E.J. Sullivan, M.T. Cronin, C.P. Holmes, and S.P.A. Fodor, Light-generated oli-gonulceotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. 91, 5022-5026 (1994). 

Agricultural Products for DNA sequence analysis and financial support. 

Cliften, P.F., et al., "Surveying Saccharomyces Genomes to Identify Functional Elements by Comparative DNA Sequence Analysis," Genome Res., 11,1175-1186 (2001). 

Once identified as a selenoprotein in this model (C. sticklandii), the need for selenium was also shown for C. sporogenes The addition of selenium to the culture medium was reported to improve the level of D-proline reductase activity as early as 1976, ° yet the first identification of the selenoprotein component of this enzyme did not occur until more recentiy in 1999 by Andreesen s group. It is quite clear now from data from these model systems, as well as from DNA sequence analysis of the grd aiiAprd operons, ° ° that Stickland reactions are common to many amino acid-fermenting clostridia. Those that are capable of proline reduction all... 

E.-B. Brodie of Brodie, S. Nicolay, M. Touchon, B. Audit, Y. d Aubenton-Carafa, C. Thermes, and A. Arneodo, From DNA sequence analysis to modelling replication in the human genome. Phys. Rev. Lett. 94, 248103 (2005). 

As the NNRTIs are structurally diverse and yet bind to RT at a common site, the similar occurrence of resistance-conferring mutations is not surprising. As a consequence, the effectiveness of other NNRTIs may be compromised by the emergence of HIV-1 variants caused by a previous NNRTI therapy (Sardana et ah, 1992). Experiments were performed in which HIV-1 strains (JR-CSF or ME) are cultured in human lymphocytes in the presence of partially inhibitory concentrations of delavirdine (Dueweke et al., 1993b). These conditions yield mutants that are 100-fold resistant. In order to determine what mutation(s) occurs, PCR (polymerase chain reaction) amplification and DNA sequence analysis of the RT coding region were applied and indicated that mutation P236L had occurred. Mutations at amino acids 181 or 183, which have been associated with resistance to other NNRTIs, are not detected. 

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. 

The limitations of genomic analysis are being addressed by proteomic analysis. DNA sequence analysis has helped to create protein product libraries and relational databases that hold promise to... 

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). 

Recio et al. (1998) also examined the lad mutagenicity and mutational spectrum in the spleen of mice exposed to butadiene in the above study. The authors reported three-and four-fold increases in the lad mutant frequency in mice exposed to 625 or 1250 ppm butadiene, respectively, compared with air control mice. DNA sequence analysis of lad mutants recovered from the spleen of mice exposed to 1250 ppm butadiene once again revealed an increase in mutations at A T base pairs (10/57, 18%) in butadiene-exposed mice compared with air control mice (3/41, 7%). In addition, an increased frequency of... 

Graves, R.J., Trueman, P, Jones, S. Green, T. (1996) DNA sequence analysis of methylene chloride induced HPRT mutations in CHO cells comparison with the mutation spectrum obtained for 1,2-dibromomethane and formaldehyde. Mutagenesis, 11, 229-233... 

Silver, S., B. P. Rosen, and T. K. Misra. 1986. DNA sequencing analysis of mercuric and arsenic operons of plasmids from gram negative and gram positive bacteria. In 5th International Symposium on the Genetics of Industrial Microorganisms. Alacevic, M., D. Hranueli, and Z. Toman (eds). pp 357-371. 

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