1.

NCBI - Nucleotide and Protein Sequences

AIM:
To retrieve nucleotide and protein sequences of given organism and to find the
chromosomal location, cell line and tissue type.

INTRODUCTION:
NCBI is a National resource for molecular biology information. It creates public databases,
develops software tools for analyzing genome data and disseminates biomedical information for
better understanding of molecular processes that affect human health. NCBI conducts research on
fundamental biomedical problems at the molecular level using mathematical and computational
methods, develops and promotes standards for databases, data deposition and exchange, and
biological nomenclature.

METHODOLOGY:
1. Access NCBI homepage using www.ncbi.nlm.nih.gov/
2. Select Nucleotide or Protein in search box.
3. Type the name of the organism/gene/protein for which the sequence has to be retrieved.
4. Click OK.
5. The NCBI entry page which contains all the information of the sequence will be
displayed. Select one. Genbank page displays the entry with accession number, locus,
function, cell line, tissue types, and chromosomal location.
6. To get the sequence in the FASTA or other formats click on the display menu and
sequence will be open in the corresponding format, which can be retrieved.

RESULT
The protein ___________ bearing gi code _________ and the sequence has been retrived in
FASTA format.

Viva Questions
1. Define database
2. Expand NCBI
3. NCBI is maintained by-------------&--------------
4. FASTA format starts with--------
5. Flat file format starts with -------------- and ends with ------------------------

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 2. SWISSPROT – Protein Data
AIM:
To retrieve data for protein ______________ from Swissprot database.

INTRODUCTION:
Swissprot is a curated Protein sequence database which strives to provide a high level of
annotation (such as the description of the function of the protein, its domain structure, post-
translational modification, variance etc,) with a minimum level of redundancy and high level of
integration with other database. The Swissprot database has been a collaborative effort of the
department of Medical Biochemistry of University of Geneva and what was then called the data
library group of European Molecular Biology Laboratory (EMBL). The founder, Amos Bairoch is
ultimately responsible for the scientific content and format of Swissprot. Uniprot KB/Swissprot is
accompanied by Uniprot/TrEMBL, contains the translation of all coding sequences (CDS) present
in DDBJ/EMBL/Genbank nucleotide sequence databases and also protein sequence extracted from
the literature or submitted to Uniprot KB/Swissprot.

METHODOLOGY:
1. Swissprot homepage was accessed using www.expasy.org/sprot
2. The name of the protein ____________ for which data has to be retrieved was entered.
A search was performed.
3. The Swissprot database displays the entry page for the protein _____________
containing entry name, primary accession number, date at which it is integrated to
Swissprot, protein name, gene name, function, properties, pfam id, prosite id, sub
cellular location, cross references and sequence information.

RESULT:-

The sequence of the protein ____________ with entry name __________ and primary accession
number ____________ was retrieved from Swissprot.

Viva Questions
1. Define protein
2. Swiss prot database is maintained by----------------
3. Swiss prot format ends with-------------
4. Expasy stands for --------
5. URL of Swissprot is-----------------------

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 3. PDB- PROTEIN DATABANK

AIM:

To retrieve structure data for protein ____________ from PDB database.

DESCRIPTION:

The Protein data bank (PDB) is the single worldwide repository of information about the 3-
dimensional structures of large biological molecules including Proteins and Nucleic acids. A
variety of information associated with each structure is available through the RCSB. PDB including
sequence details, atomic coordinates, crystallization conditions, 3D structure neighbors, derived
geometric data, structure factors, 3D images, and a variety of links to other resources. Using the
PDB we can download protein coordinate file, display the molecule and perform structural analysis.

METHODOLOGY:

1. PDB homepage was accessed using the URL http://www.rcsb.org/pdb

2. Type the protein “__________” in search column.
3. A search is performed.
4. The PDB database displays the entry page for globulin containing information about the
title, release date, experimental method, parameters, unit-cell information, fragment,
source, classification, sequence details, SCOP, CATH, Pfam links and resolution etc,

RESULT:

The protein sequence of bearing PDB code which is obtained from

NMR a novel studies with tittle was retrived from PDB.

Viva Questions
1. Define protein
2. PDB stands for----------------
3. Techniques used for predicting the structures of biomolecules-------------
4. Expasy stands for --------
5. URL of PDB is-----------------------

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 4. CATH – Protein Data

AIM: To retrieve structure data for protein ___________ from CATH database.

DESCRIPTION:

CATH is a hierarchical classification of protein domain structures with clusters of proteins

at four major levels, Class (C), Architecture (A), Topology (T), Homologous superfamily (H).
Class is derived from Secondary structure content is assigned for more than 90% of protein
structures automatically. Architecture describes the orientation of the secondary structures
independent of connectivity, as currently assigned manually, the topology level clustered structures
into fold groups according to their topological connections and numbers of Secondary structures.
The homologous superfamily clusters proteins with higher Secondary structures and function. The
assignments of structures to fold groups and homologous super families are made by sequence and
structure comparison.

METHODOLOGY:

1. The CATH homepage was accessed using the URL www.cathdb.info

2. The name of the protein “________” was entered and a search was performed.
3. A page displays a list of domains, chains and PDB code.
4. When clicked on the domain link, a page is displayed containing information about
the class, architecture, topology, and homologous superfamily for ___________.

RESULT:

The CATH data of the protein __________ were retrieved as following:

Domain : _________
C : Class : _________
A : Architecture : __________
T : Topology : __________
H : Homology : ___________
Viva Questions
1. CATH stands for
2. SCOP stands for----------------
3. All enzymes are proteins (T/F)
4. All proteins are enzymes (T/F)
5. URL of CATH is-----------------------

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 5.Local Alignment with Smith Waterman Tool.

Aim:
To perform local alignment for coronin and action of homosapiens by Waterman Tool.

Procedure:

1.The FASTA Format of protein sequence of coronin of Homosapiens and action of Homosapiens
is obtained by accessing NCBI.

2.Logged into Waterman Tool available at http://www.ebi.ac.uk/emboss/align.

3.Post the two sequences in appropriate input boxes in Waterman Tool.

4.Submit the sequence by clicking the red button.

5.Results were displayed as soon as the task completes.

RESULT:
A similar search was performed for the protein coronin and action of Homosapiens using emboss
local alignment tool and the results were retrieved.

Viva Questions
1. Define Dynamic programming
2. Algorithm used for local alignment of sequences----------------
3. Online tool used for local alignment of sequences
4. Define pairwise alignment
5. Mention the two types of alignment

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 6.GLOBAL ALIGNMENT NEEDLEMAN WUNCH TOOL

Aim:
To perform global alignment for keratin and coronin of Homosapiens by using Needleman Tool.

Procedure:

1.The FASTA format of protein sequence of keratin of Homosapiens and coronin of homosapiens
is obtained by accessing NCBI.

2.Log into Needleman wunch Tool available at http://www.ebi.ac.uk/emboss/align.

3.Paste the two sequences in appropriate inboxes in Needleman Tool

4.Submit the sequences by clicking the run button.

5.Results were displayed as soon as the task complete.

RESULT:
The proteins keratin and coronin of Homosapiens were performed using emboss Global alignment
tool and result was retrieved.

Viva Questions
1. Define Dynamic programming
2. Algorithm used for global alignment of sequences----------------
3. Online tool used for global alignment of sequences
4. Define pairwise alignment
5. Mention the two types of alignment

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 7.DOT MATCHER

Aim:

To draw a threshold Dot Prot for two sequences limphactin and fibrinogen.

Procedure:

1. The two nucleotide sequences in FASTA format were obtained by accessing nucleotide
sequence database.
2. Log into http://emboss.bioinformatics.ni/cgi-binlemboss/dotmatcher.
3. Paste the respective sequences in the given input boxes
4. Click on run dot matcher.
5. Results were displayed as soon as the task completes.

RESULT:
Dot Prot was obtained from the nucleotide sequences limphactin and fibrinogen.

Viva Questions
1. Define pairwise alignment
2. Fasta format starts with----------
3. EMBOSS tool is used for alignment of ------------------
4. Dot matcher is used for------------
5. URL of Dot matcher

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 Sequence Similarity Search Tools
8. BLAST N
Aim
To find the similar sequence to a given nucleotide sequence query from nucleotide database.
Description:
The comparison of nucleotide or protein sequence from the same or different organism is a very
powerful tool in molecular biology. By finding similarity between sequences one can infer the
function of newly sequenced genes, predict new membranes of gene families and explore
evolutionary relationships. Basic local alignment search tool (BLAST) is the tool most frequently
used for calculating sequence similarity.
Methodology:
1. BLAST can be accessed from the URL: www.ncbi.nlm.nih.gov/blast
2. BLAST N was selected for nucleotide – nucleotide similarity search.
3. Paste the “_________” nucleotide sequence in FASTA format in BLAST N page.
4. The options can be used to set the length of the sequence.
5. The database to be included for the search was selected from the pull down menu.
6. ‘BLAST’ button was clicked to run the search.
7. A page was displayed showing the ID.NO and appropriate wait time. Format button was
clicked for result page.
RESULT :-
A BLAST N search was performed for nucleotide ___________ with gi code _________ and
_____ BLAST hits were retrieved.

Viva questions
1.BlastN stands for---------
2.Nature of query in Blast N------------
3.Nature of database in Blast N------------
4.Blast program was designed by-----------
5.Online tool used for evolutionary studies-------------

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 9. BLAST P
Aim:
To find the similar sequence to the given protein sequence query using BLAST P

Description:
Standard protein – protein BLAST (BLAST P) is used for both identifying a query amino
acid sequence and for finding similar sequences in protein databases. Like other BLAST
programs, BLAST P is designed to find local regions of similarity. When sequence similarity
spans the whole sequence, BLAST P will also report a global alignment, which is the preferred
result for protein identification purposes.

Methodology:
1. BLAST can be accessed from the URL: www.ncbi.nlm.nih.gov/blast
2. BLAST P was selected for protein – protein similarity search.
3. Enter __________ nucleotide sequence in FASTA format in BLAST P page.
4. The options can be used to set the length of the sequence.
5. The database to be included for the search was selected from the pull down menu.
6. ‘BLAST’ button was clicked to run the search.
7. A page was displayed showing the ID.NO and appropriate wait time. Format button was
clicked for result page.
RESULT:
A BLAST P search was performed for the protein __________ with gi code _________ and
______ hits were retrieved.
Viva questions
1.BlastP stands for---------
2.Nature of query in Blast P------------
3.Nature of database in Blast P------------
4.Blast program was designed by-----------
5.Online tool used for evolutionary studies-------------

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 10. BLAST X
Aim:
To find the similar sequence for a given nucleotide query using BLAST X.
Description:
Translated query Vs protein database (BLAST X) is useful for finding similar proteins to those
encoded by a nucleotide query. Translated query BLAST services are useful when trying to find
homologous protein to a nucleotide coding region. BLAST X compares translational products
of the nucleotide query sequence to a protein to a protein database. Because BLAST X
translates the query sequence in all six reading frames and provides combined significance
statistics for hits to different frames, it is particularly useful when the reading frames of the
query sequence is unknown or it contains errors that may lead to frame shifts or other coding
errors. Thus BLAST X is often the first analysis performed with a newly determined nucleotide
sequence and is used when extensively analyzing EST sequences. This search is more sensitive
than nucleotide blast since comparison is performed at the level.
Methodology:
1. BLAST can be accessed from the URL: www.ncbi.nlm.nih.gov/blast
2. BLAST X was selected which was used for translated query and protein database similarity
search.
3. Paste __________ nucleotide sequence in FASTA format in BLAST page.
4. The options can be used to set the length of the sequence.
5. The database to be included for the search was selected from the pull down menu.
6. ‘BLAST’ button was clicked to run the search.
7. A page was displayed showing the ID.NO and appropriate wait time. Format button was
clicked for result page.
RESULT:
A BLAST X search was performed for the nucleotide sequence of _________ protein with gi code
________ and _______ hits were retrieved.

Viva questions
1.BlastX stands for---------
2.Nature of query in Blast X------------
3.Nature of database in BlastX------------
4.Blast program was designed by-----------
5.Online tool used for evolutionary studies-------------

10
 11. T BLAST N
Aim:
To find the similar sequences for a given protein query using t blast n.
Description:
Protein query Vs translated database (t blast n) is useful for finding protein homolog is annotated
nucleotide data. A t blast n search allows you to compare a protein sequence to the six reading
frame translations of a nucleotide database. It can be a very productive way of finding homologous
protein coding regions in an annotated nucleotide sequence such as expressed sequence tags
(EST’s) and draft genome records (HTG), located in the BLAST databases EST and HTG s
respectively.
Methodology:
1. BLAST can be accessed from the URL: www.ncbi.nlm.nih.gov/blast
2. T BLAST N was selected which was a similarity search between a protein query and
translated database.
3. Paste _________ protein sequence in FASTA format in BLAST page.
4. The options can be used to set the length of the sequence.
5. The database to be included for the search was selected from the pull down menu.
6. ‘BLAST’ button was clicked to run the search.
7. A page was displayed showing the ID.NO and appropriate wait time. Format button was
clicked for result page.
RESULT:

A T BLAST N search was performed for protein sequence of _______ with gi code _______ and
______ hits were retrieved.

Viva questions
1. TBlastN stands for---------
2.Nature of query in T Blast N------------
3.Nature of database in T Blast N------------
4.Blast program was designed by-----------
5.Online tool used for evolutionary studies-------------

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 12. CLUSTAL W

AIM:
To perform multiple sequence alignment of nucleotide/protein sequences and to build a
phylogram for the given set of sequences using CLUSTAL W.
INTRODUCTION:
Multiple alignments of protein sequences are important tools in studying sequences. The basic
information they provide is identification of conserved sequence regions. This is very useful in
designing experiments to test and modify the function of specific proteins and in identifying a new
member of protein families. CLUSTAL W is a general purpose multiple sequence alignment
program for DNA or proteins. It produces biologically meaningful multiple sequence alignment of
divergent sequences. It calculates the best match for the selected identities, similarities and
differences can be seen. Evolutionary relationship can be seen via viewing cladograms or
phylograms.
METHODOLOGY:
1. CLUSTAL W homepage was accessed using www.ebi.ac.uk/clustalw
2. The alignment title, results, alignment score type, matrix, gap open, output order, tree type
etc was selected.
3. The nucleotide/protein sequences were entered.
4. ‘Run’ button was clicked.
5. A result page was displayed showing the multiple alignment of the nucleotide
sequences/protein sequences along with the phylogram.
RESULT :
A similarity search for protein sequences of __________ (gi codes _____ ) was performed using
CLUSTAL W tool, and results were visualized for alignment file, cladograms, phylograms and
Jalview were downloaded.

Viva questions
1.Define multiple sequence alignment
2.Clustal W is used for------------
3.Clustal Omega is used for------------
4.Muscle is used for-----------
5.Online tool used for multiple sequence alignment-------------

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 13.PROTEIN SECONDARY STRUCTURE PRODUCTION

A.GOR-IV

Aim:
To predict the secondary structure of thrombin protein using GOR.

Procedure:
1. The protein sequence was retrieved from NCBI database in FASTA format.
2. Log into http://npsa-pbil.bcp.fr/egi-bin/npsa-automat.pl?page=npsa-gor4.html
3. The protein sequence was pasted in input box.
4. Submit button was clicked inorder to submit protein.
5. The results were obtained after the completion of task.

RESULT:
Results were obtained representing secondary structure elements of thrombin with their single letter
codes such as α-helix, β-sheets, coil of α-helix.

Viva questions
1.Define protein---------
2.Peptide is formed between------------ &--------------
3.The two ends of a protein are------------&-------------------
4.Secondary structure of protein consists of -----------
5.Online tool used for Secondary structure prediction-------------

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 B. JPRED

Aim:
To predict the secondary structure of thrombin by using JPRED software.

Procedure:
1. The protein sequence was retrieved from NCBI database in FASTA format.
2. Log into http://www.compbio.dundee.ac.uk
3. The sequence was pasted in input box.
4. The sequence was submitted by clicking make prediction button.
5. The result was obtained after the completion of the task.

RESULT:
The results were obtained representing secondary structure elements with their single letter codes
such as α-helix, β-sheets.

Viva questions
1.Define protein---------
2.Peptide is formed between------------ &--------------
3.The two ends of a protein are------------&-------------------
4.Secondary structure of protein consists of -----------
5.Online tool used for Secondary structure prediction-------------

14
 C.SOPMA

Aim:

To predict the secondary structure of thrombin protein using SOPMA.

Procedure:

1. The protein sequence was retrieved from NCBI database in FASTA format.
2. Log into http://npjaupbil.ihcp.fr/eginbin/npsa.automat.pl?page=/NPSA/npsa-sopma.html
3. Post the protein sequence in input box.
4. Submit the sequence by clicking in submit button.
5. The results were obtained after completing the task.

RESULT:
Results were obtained representing secondary structure elements with their single letter codes such
as α-helix, β-sheets.

Viva questions
1.Define protein---------
2.Peptide is formed between------------ &--------------
3.The two ends of a protein are------------&-------------------
4.Secondary structure of protein consists of -----------
5.Online tool used for Secondary structure prediction-------------

15
 D.TMPRED
Aim:

To predict the trans-membrane region and orientation of thrombin protein using TMPRED.

Procedure:

1. The protein sequence was retrieved from NCBI database in FASTA format.
2. The website http://www.ch.embnet.org/software/TMPRED from htmal was logged onto
perform secondary structure prediction.
3. The protein sequence was pasted in input box.
4. Sequence was submitted by clicking on submit button.
5. Result was obtained after the completion of task.

RESULT:
Transmembrane region for protein thrombin was It is membrane- spaning region prediction.

Viva questions
1.Define protein---------
2.Peptide is formed between------------ &--------------
3.The two ends of a protein are------------&-------------------
4.Secondary structure of protein consists of -----------
5.Online tool used for Secondary structure prediction-------------

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 14.STRUCTURE VISUALIZATION TOOL : RASMOL

Aim:
To visualise and determine the structure of protein mucin using RASMOL.

Introduction:

RASMOL is a free interactive molecular graphics viewer intend for the visualization of
protein nucleic acid and small molecules. The program read in the 3D coordinate for
molecule using the PDB file format. Currently available representation includes depth
covered sticks, space filling sphere solid and strand bimolecular ribbons, atoms and dot
surface. It displays the molecules in various representation and allows one to rotate the
molecule interactively. The name RASMOL came from display Raster is type of computer
especially useful for solid surface.

Methodology:

1. To run RASMOL, two windows should be opened. A graphics window and a common
tiny window.
2. To visualize, the molecule opened the atom coordination file in PDB format in Raswin
by selecting the file in the RASMOL title bar and then opened.
3. Choose the PDB file and select OK.
4. To open another PDB file, the command file must be close. To do this, select close
under file in the RASMOL title bar and then to open multiple file (i.e. each can view up
to 5 molecules) simultaneously File/open (For each file, without closing the previous
file) from within RASMOL. From within NETSCAPE, just click on another RASMOL
viewable molecule.

17
 5. To close a file, File close when the molecule is selected.

Rotation Left mouse button (Click & Hold).

Translation Right mouse button (Click & Hold).
Zoom <AH> <Shift> & left mouse button.
Z- Rotation <Right> <Shift> & right mouse button.

To change different representation i.e stick, ribbon etc. Display stick.

To determine angle: Click appropriate icon in the molecular window (the secondary
window).
Click once on the appropriate number of individuals atoms (with angles, clicking on
atoms must be in appropriate order).
To rotate bond: Click on rotate angle icon in the molecules wndow (the secondary window).
Click on second atom
Click on rotate angle icon in the molecule window again.
Click on third atom
Use mouse button to rotate
To colour by protein secondary structure:
Alpha helix Magenta
Beta sheets Yellow
Turns Pale blue
All other residues White

To colour by residue type (i.e each type of residue is coloured by specified

colour).
To colour by atom type (below the default colours.)
Carbon Grey
Hydrogen While
Oxygen Red
Nitrogen Blue
Sulphur Yellow
Iron Yellow

Result:
Thus the structure of _____________________ from crystal structure of
______________________________.

18
 Viva questions
1.Rasmol is used for---------
2.3D structures of biomolecules are visualized by ------------&-------------
3.Ramachandran plot is between ------------&----------------
4.Structural database-----------
5.Online tool used for visualization-------------

15.MOLECULAR EVOLUTIONARY GENETIC STUDIES BY MEGA

AIM: -

To construct different phylogenetic trees and evolutionary distance matrices using MEGA5.0 total.

INTRODUCTION: -

The molecular evolutionary genetics analysis (MEGA) software is a desktop application designed
for comparative analysis of homologous gene sequences either from multigene families or from
different species with a special emphasis on inferring evolution. In addition to tools for statistical
analysis of data, MEGA provides many convenient facilities for the assembly of sequence data sets
from files, and it includes tools for visual presentation of results obtained in the form of interactive
phylogenetic trees and evolutionary distance matrices.

PROCEDURE: -

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1) MEGA was started by selecting the windows start menu 🡪 programs🡪MEGA5 – click on the
MEGA icon.
2) Click on the file and select open a file🡪examples🡪select required MEGA file.
3) When data file is active, the input distance data viewer is launched to display contents of data
file.
4) Clicked on distance🡪compute pair wise distance command.
5) After that clicked on model🡪compute amino acid composition to display amino acids present in
data file.
6) Now constructed different phylogenetic trees using distance matrix.

A. Maximum Likelihood Method: -

1) Selected the phylogeny🡪construct phylogeny🡪maximum likelihood command🡪clicked ok.

2) Clicked”COMPUTE” to accept the defaults for rest of options and begin the computations. A
program indicator will appear briefly before tree display in tree explorer.
3) Changed the branch style by using view🡪 tree/branch style command.
4) Selected the view🡪topology only command from tree explorer menu to display branching
pattern.
5) Clicked on view🡪 show/hide🡪branch length menu to display branch lengths on screen.

B. Neighbour Joining tree method: -

1) Selected the phylogeny🡪construct phylogeny🡪neighbour joining command to display the

analysis preferences dialog box.
2) In the options summary tab, clicked on models pull-down & then selected the p-distance option.
3) Clicked “compute” to accept the defaults for the rest of options and began the computations. A
progress indicator will appear briefly before the tree displays in the tree explorer.
4) Clicked on view🡪show/hide🡪branch lengths menu to display branch lengths on screen.

C. Minimum Evolution method: -

1) Selected phylogeny🡪constructs phylogeny🡪minimum evolution command to display analysis

preference box.
2) Clicked “compute” to accept the defaults for the rest of options and begun the computations.
3) Clicked on view🡪show/hide🡪branch lengths menu to display branch lengths on the screen.

D. UPGMA method: -

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1)Selected phylogeny🡪construct phylogeny🡪UPGMA command to display the analysis preference
dialog box.
2)Clicked “compute” button to accept the defaults for the other options and begun the calculations.
3)Clicked on view🡪show/hide🡪branch length menu to display branch lengths on screen.

E. Maximum parsimony method:-

1)Selected phylogeny🡪construct phylogeny🡪maximum parsimony command to display analysis

preference dialog box.
2)Clicked “compute” button to accept the defaults for other option & begun the computations. A
progress window will appear briefly and the tree will displayed in tree explorer.
3)Clicked on view🡪show/hide🡪branch length menu to display branch lengths on screen.

RESULT: -

Different phylogenetic trees were constructed by using MEGA5.0.

Viva questions
1.MEGA stands for---------
2.Expand UPGMA ------------
3.Distance based method---------
4.Character based methods-----------
5.Online tool used for evolutionary studies-------------

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