Nucleotide Sequence Analysis

Complementary DNAs (cDNA) are DNA copies of mRNAs. Reverse transcriptase is the RNA-directed DNA polymerase that synthesizes DNA strand, using purified mRNA as the template. DNA polymerase is then used to copy the DNA strand forming a double-stranded cDNA, which is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries (Cowell and Austin, 1997). Annotated human cDNA sequences can be accessed from HUNT at http // www.hri.co.jp/HUNT (Yudate, 2001). 

Sequence data can be analyzed for (a) Sequence characteristics by knowledge-based sequence analysis, (b) Similarity search by pairwise sequence comparison, (c) Multiple sequence alignment, (d) Sequence motif discovery in multiple alignment, and (e) phylogenetic inference. 

The nucleotide sequences can be retrieved from one of the three IC (International Collaboration) nucleotide sequence repositories/databases GenBank, EMBL Nucleotide Sequence Database, and DNA Data Bank of Japan (DDBJ). The retrieval can be conducted via accession numbers or keywords. Keynet (http // www.ba.cnr.it/keynet.html) is a tree browsing database of keywords extracted from 

All of the three IC centers also provide facilities for sequence similarity search and alignment. The widely used database search algorithms are FASTA (Lipman and Pearson, 1985) at http //www.nbrf.georgetown.edu/pirwww/search/fasta.html and BLAST (Altschul et al., 1990) at http //www.ncbi.nlm.nih.gov/BLAST/. For BLAST 

The catalog of synthetic oligonucleotides which are proven useful as PCR primers or gene probes can be downloaded from National Cancer Research Institute 

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Johnson GR, RH Olsen (1995) Nucleotide sequence analysis of genes encoding a toluene/benzene-2-mono-oxygenase from Pseudomonas sp. strain JS150. Appl Environ Microbiol 61 3336-3346. 

J. Morris, D. F. Donnelly, E. O Neill, F. McConnell, and F. O Gara, F. Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp. strain Ml 14. Mol. Gen. Genet. 242 9 (1994). J. M. Raaijmakers, W. Bitter, H. L. M. Punte, P. A. H. M. Bakker, P. J. Weisbeek, and B. Schippers, Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas piitida WCS358 in natural environments. Appl. Environ. Microbiol. 60 1184 (1994). 

Huber M, Garfinkel L, Gitler C, Mirelman D, Revel M, Rozenblatt S (1988) Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene. Mol Biochem Parasitol 31 27-33... 

Ellis JE, Yarlett N, Cole D, Humphreys MJ, Lloyd D (1994) Antioxidant defenses in the microaerophilic protozoan Trichomonas vaginalis—comparison of metronidazole-resistant and sensitive strains. Microbiol-SGM 140 2489-2494 Haggoud A, Reysset G, Azeddoug H, Sebald M (1994) Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacterioides strains and of a new insertion sequence upstream of the two genes. Antimicrob Agents Chemother 38 1047-1051... 

Soon after the discovery of RNase Ti, it was suggested (70, 71) that it would become an important tool for the elucidation of nucleotide sequence in RNA. Indeed, since 1962 several workers have tried to use the enzyme for the nucleotide sequence analysis of RNA, especially in highly purified specific tRNA s. Finally, the brilliant research of Holley and his associates in 1965 resulted in the first elucidation of the complete nucleotide sequence of an RNA, alanine specific yeast tRNA, using RNase Ti as a main tool (29). Since then many successful elucidations of nucleotide sequence of various RNA s, using RNase Ti as a main tool, followed, and now the enzyme is well-known as an essential tool for the structural analysis of RNA. 

Although, because of its complicated specificity, it is not easy to use the enzyme with a probable expectation for the nucleotide sequence analysis of RNA, sometimes it has been used with success (115, 116). It may be useful especially to digest the large fragments produced by RNase T,. In this case, it usually splits 3 -adenylyl and 3 -uridylyl bonds but not 3 -cytidylyl bonds. 

Whittier, R.F., Dean, D.A. Rogers, J.C. (1987). Nucleotide sequence analysis of a-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells. Nucleic Acids Research 15, 2515-35. 

Enzymes that are structurally related to the eukaryotic V-ATPase are also found in certain eubacteria (Speelmans etal., 1994 Takase etal., 1994 Yokoyama etal., 1990). Based on nucleotide sequence analysis, it is believed that these bacterial V-like ATPases have been introduced into the eubacteria via horizontal gene transfer from Archaea (Hilario and Gogarten, 1993, 1998). The subunit composition of the bacterial V-like ATPase is indeed more similar to the archaeal A-ATPase than to the eukaryotic V-ATPase, and we will therefore treat the bacterial V-ATPase—like enzyme together with the archaeal A-ATPase (see below). In the following, we will use the name V-ATPase only for the eukaryotic enzyme, and we will call the bacterial enzyme the A/V-type ATPase as suggested by Hilario and Gogarten (1998). 

Neal RJ, Chater KF (1987) Nucleotide sequence analysis reveals similarities between proteins determining methylenomydn A resistance in Streptomyces and tetracycline resistance in eu-bacteria. Gene 58 229-241. 

A Arisawa, H Tsunekawa, K Okamura, R Okamoto. Nucleotide sequence analysis of the carbomycin biosynthetic genes including the 3-O-acyltransferase gene from Streptomyces thermotolerans. Biosci Biotechnol Biochem 59 582-588, 1995. 

Similarity Searching (BLAST) Nucleotide Sequence Analysis Protein Sequence Analysis Molecular Structure Analysis t Genome Analysis Gene Expression... 

The SPL (search for potential splice sites) tool of the Sanger Centre predicts splice sites of an input query sequence. On the nucleotide sequence analysis page, paste the query sequence, enter the sequence name, select SPL tool, and click the... 

Sanger Centre, home Sanger Centre, Nucleotide Sequence Analysis Sanger Centre,... 

Fig. 3.10. Autoradiograph of a sequencing gel prepared using the Maat and Smith procedure. The sequence shown is that derived from a 440 nucleotide-long fragment from a Hinfl digest of a 5 -end labelled HirtdUl fragment of adenovirus type 5 DNA. Samples from each base-specific reaction mixture were loaded every 2 hours (runs I, II, III, and IV). Electrophoresis was carried out at a constant current of 30 mA. Nucleotide sequence analysis of the complementary DNA strand revealed one mistake in the sequence as written. At position 2870 (in run III) two C s should be read instead of one. The zone of compression responsible for this error is not very apparent and emphasizes the importance of sequencing both DNA strands.

Mills, D.R. and F.R. Kramer, 1979. Structure-independent nucleotide sequence analysis. Proc. Natl. Acad. Sci. USA 76, 2232. 

Stalker, D.M., Malyi, L.D., and McBride, K.E. 1998. Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. Journal of Biological Chemistry, 263 6310-4. 

Mittler, R., and Zilinskas, B. A., 1991a, Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase, FEES Lett. 289 257n259. 

Cheneby, D., Hallet, S., Mondon, A., Martin-Laurent, F., Germon, J. C., and Philippot, L. (2003). Genetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis. Microb. Ecol 46, 113-121. 

Brown EG, Furesz J, Dimock K, Yarosh W, Contreras G. Nucleotide sequence analysis of Urabe mumps vaccine strain that caused meningitis in vaccine recipients. Vaccine 1991 9(ll) 840-2. 

In an outbreak in Egypt during 1988-93, 32 cases of poho were associated with vaccine-derived poliovirus type 2 (27). Nucleotide sequence analysis performed during 1999 showed that all isolates were related (93-96% similarity) to the OPV 2 vaccine strain. The isolates were not related (less than 81% similarity) to the wild poliovirus type 2 that had been indigenous in Egypt. OPV was probably low in the affected communities. 

A variety of powerful methods is available for fractionating short oligonucleotides. Work on nucleotide sequence analysis of small RNA molecules (n=80 or 120) and more recently viral RNA molecules (n= 3000) and even ribosomal precursor RNAs (up to 45 s) (Holley et al. 1965a Brownlee 1972 Maden et al. 1972) has stimulated the development of these procedures. They can also be used for synthetic oligonucleotides (e.g. Hachman and Khorana, 1969). The methods depend largely on chromatography and electrophoresis on filter papers, diethylaminoethyl(DEAE) paper, cellulose acetate, thin layers of cellulose or polyethyleneimine (PEI)-cellulose, or columns. 

Other mixtures of oligonucleotides must be fractionated in larger quantities or are too complex to be separated by traditional paper chromatography and electrophoresis. Large quantities (> 1 mg) of material arise in preparative procedures, in nucleotide sequence analysis of nucleic acids which are not radioactively labelled, and in mixtures where the components of interest are present in very small proportions. Column chromatography is widely used in these cases. 

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