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In-Depth Information
thiocyanate (GuSCN) or guanidinium hydrochloride (GuHCl) buffer, which desta-
bilizes nucleases. By using a silica matrix, which binds nucleic acids, specimens
can yield DNA for subsequent PCR amplification yet are suitable for examination
of their morphology ( Jeyaprakash and Hoy 2010 ).
The sequencing outcome depends upon the purity of the DNA used and the
fidelity of the sequencing procedures. It is important to sequence both strands
to detect errors. Once sequence information has been obtained, analysis can
provide several types of information, including possible structure, function,
and characteristics of the protein. The similarity of the sequences to sequences
obtained from other organisms can be compared. The tasks of collating, assem-
bling, and correcting the sequence data are usually performed with the help of
a variety of computer programs ( Gribskov and Devereux 1991, Eernisse 1998,
Fortna and Gardiner 2001, Mount 2001, Felsenstein 2004, Knowles and Kubatko
2010, Hall 2011 ).
Analysis of sequence data involves several steps ( Figure 12.4 ). Figure 12.5A
shows the form in which Sanger sequence data is obtained from a sequencing
laboratory. Figure 12.5B shows the data after they have been analyzed to show
the open reading frame (ORF) of the coding strand (the line with the •), the
codons, and the region at the end of the sequence that represents the vector
DNA, which should be excluded from the subsequent analysis.
12.6.5 Sequence Comparisons with BLAST
Once sequences have been obtained, the scientist usually wishes to compare
them with other sequences in the databases ( Figure 12.4 ). Genetic sequences are
stored in three major databases: GenBank in the United States, the EMBL Data
Library in Europe, and the DNA Data Bank of Japan. The amount of sequence
information available has grown exponentially and continues to accumulate at
an ever-faster pace. The importance of submitting DNA sequence data to public
databases is recognized and most journals require that sequences be submitted
before, or simultaneous with, publication.
Sequences can be obtained from these databases using a computer pro-
gram called Basic Local Alignment Search Tool ( BLAST ). BLAST (and its sub-
types) is one of the most widely used tools in phylogenetic analysis. BLAST is
used to search large databases of DNA (or amino-acid) sequences, returning
sequences that have regions of similarity to the sequence of interest provided by
the user (query sequence) ( Fortna and Gardiner 2001, Gibson and Muse 2002 ).
The goal is to find regions in sequence pairs that have high levels of similar-
ity. The results of a BLAST search orders the sequences and provides an e-value.
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