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 BASIC LOCAL ALIGNMENT SEARCH
TOOL (BLAST)
 The Basic Local Alignment Search Tool (BLAST) is an algorithm and program that finds
the region of local similarity between the sequences by the comparison of the primary
biological sequence information of amino acids found in proteins or the nucleotides of
DNA or RNA sequences.
 The current BLAST command-line applications were made available to the public in late
1997, and BLAST is part of the NCBI toolkit.
 It is a search program for sequence similarity that can quickly search a sequence database
to match with the query/unknown sequences.
 There are different sub-types of the BLAST that is used for comparing all combination of
nucleotide and protein query sequences against a nucleotide or proteins database.
 It can be used to observe the functional and evolutionary relationship between sequences as
well as help identify members of a gene family.
 The initial 1997 BLAST tool lacked various features, but the present-day BLAST is able to
handle databases with more than two billion letters.
USES OF BLAST

 It is used for searching the database like:

i. Having a new isolated sequence but have no idea of what it might be of.

ii. Identification of the name and function.

 To design PCR primers.

 Checking the PCR primers for specificity.

 APPLICATION OF BLAST
1. DNA mapping: BLAST plays an important role to compare the chromosomal positions of the
sequence of interest to reveal the sequences in the database when looking for the specific gene at an
unknown location and working with the known species. Thus, BLAST helps in the identification
and mapping of the gene between the known and unknown species.

2. Domains location: BLAST helps to identify and locate the domains in the protein sequences of
interest.

3. Comparison: while comparing the sequences between the two different or similar species, BLAST
is used and thus it helps to identify the similar genes present or the functions between the species.

4. Identification of species: BLAST can also be used for the identification of the species by the
comparison of the sequences of the DNA between the different organisms.

5. Establishing phylogeny: after the alignment of the sequences using BLAST, one can identify the
result and observe the phylogenetic relationships between the known and unknown species.
 ACCESS TO BLAST

 https://blast.ncbi.nlm.nih.gov/Blast.cgi
 FOUR TYPES OF BLAST

1. Nucleotide BLAST (BLASTn)

 This tool helps to compare one or more nucleotide sequences to reference sequences or a
database of nucleotide sequences. It is used during the determination of the evolutionary
relationship among different organisms. 
 For this, we have to enter into the NCBI website and search for the Nucleotide BLAST
option. After this, add the accession number of the reference sequences and the query
sequences. Then, for comparing sequences, find the box called align two or more
sequences under the query sequence box. Finally, click on the BLAST options leaving
other settings to their default options.
2. Protein BLAST (BLASTp)
 One or more protein sequences are compared to subject protein sequences or a database of
protein sequences by this type of BLAST search. It is used for the identification of protein
sequences.
3. BLASTx (translated nucleotide sequence searched against
protein sequence)
 BLASTx compares a nucleotide query sequence that is translated into protein sequences against the
protein sequence database. BLASTx is particularly important when the reading frame of the query
sequence is unknown, or it contains errors that may lead to frameshift or other coding errors because
it translates the query sequence in all six reading frames and provides a combined statistical
significance for hits to different frames. With a newly determined sequence, BLASTx is often the
first analysis that is performed.

4. tBLASTn (protein sequence searched against translated

nucleotide sequences)
 A query protein sequence is compared against the six-frame translation of a database of nucleotide
sequences in this type of BLAST search tool. Homologous protein-coding regions in unannotated
nucleotide sequences like expressed sequence tags (ESTs) and draft genome records (HTG), located
in the BLAST database est and htgs, respectively, can be found by tBLASTn. A short single-read
cDNA sequence is called ESTs, which consists of the largest pool of the sequenced data for many
organisms and also consists of proportions of transcripts from many uncharacterized genes. There is
no corresponding protein translation in the BLAST protein datasets since ESTs do not have
annotated coding sequences. Thus, tBLASTn is only the potential way to search these potential
coding regions at the protein level. Another large source of unannotated coding regions is the HTG
sequence, a draft sequence from various genome projects or large genomic clones.
ACCESS TO BLAST AND HOMEPAGE
1. NUCLEOTIDE BLAST (BLASTN)

CLICK ON IT
YOU WILL FIND THREE SEARCH BARS

1.Enter Query Sequence

2.Choose Search Set
3.Program Selection
 TYPES
SEARCH ENGINE
COPY FASTA SEQUENCE
1. ENTER QUERY SEQUENCE
2. CHOOSE SEARCH SET

If you want to exclude

uncultured sequences,
select this option

If you have no information, keep the settings

at default

 In the “Choose Search Set” selection box, click “Standard databases”.

 In the drop-down list choose “Nucleotide collection (nr/nt)”.
 Leave all other fields in their default state.
3. PROGRAM SELECTION

The most important one

CLICK THE “BLAST” BUTTON TO
INITIATE A BLAST SEARCH.

OR

You can also select

this option