Sequence analysis Practical
INTRODUCTION
Hox proteins are involved in the regulation of development (morphogenesis) and have
been identified in a diverse group of organisms, such as animals, fungi and plants.
Hox proteins are known to contain a characteristic signature.
The goals of this practical are to:
1. retrieve a protein from Genbank using an accession number.
2. Identify a homolog in another species using BLAST
3. Align the two sequence sequences using DOTMATCHER
(http://sf01.bic.nus.edu.sg/EMBOSS/)
4. Identify homologs in additional species
5. Align all sequences using ClustalW
1. Extract the human Hox A4 (NP_002132) protein sequence in FASTA format from
NCBI (http://www.ncbi.nlm.nih.gov/) and save it to a text file on your desktop.
2. Use the NCBI BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify
homologs of Hox A4 in mouse, sea-bass (Morone saxatilis) and horn shark
(Heterodontus francisci). Add these sequences to your protein text file
Organism
% identity between A4
orthologs
E-values
Human – Mouse
Human – Sea-bass
Human – Horn shark
3. Generate a dot-plot for human and shark Hox A4 proteins by aligning these
sequences using the DOTMATCHER software of EMBOSS
((http://sf01.bic.nus.edu.sg/EMBOSS/)
3.1 How do you interpret the long diagonal line in the top right corner of the matrix?
(remember to copy the dotplot into your report).
4. Using ClustalX, generate a multiple sequence alignment of all four Hox A4
proteins.
4.1 What region is conserved in all species …………………(amino acid position)
4.2 What is the corresponding exon number ………
4.3 Save the alignment in a textfile. How is identity indicated in the alignment?
5. Align seq1 and seq2 using the DOTMATCHER software of EMBOSS
((http://sf01.bic.nus.edu.sg/EMBOSS/)
5.1 What scenario would explain the dot-plot pattern that you observe?
5.2 Modify the window size to 100. What is the effect on the dot-plot?
>seq1
CACTCGATGCAGGCGCTGTCCTGGCGCAAGCTCTACTTAAGCCGCGCCAAGCT
CAAAGCTTCCAGCCGGACCTCGGCTCTGCTCTCCGGCTTCGCCATGGTAGCGA
TGGTGGAAGTCCAGCTGGACACAGACCATGACTACCCACCAGGGTTGCTCATC
GTCTTTAGTGCCTGCACCACAGTGCTAGTGGCCGTGCACCTGTTTGCCCTCATG
ATCAGCACCTGCATCCTGCCCAACATCGAGGCTGTGAGCAACGTCCACAACCT
CAACTCGGTCAAAGAGTCACCCCACGAGCGCATGCATCGCCACATCGAGCTGG
CCTGGGCCTTCTCCACGGTCATCGGGACGCT
>seq2
CACTCGATGCAGGCGCTGTCCTGGCGCAAGCTCTACTTAAGCCGCGCCAAGCT
CAAAGCTTCCAGCCGGACCTCGGCTCTGCTCTCCGGCTTCGCCATGGTAGCGA
TGGTGGAAGTCCAGCTGGACACAGACCATGACTAATGATCAGCACCTGCATCC
TGCCCAACATCGAGGCTGTGAGCAACGTCCACAACCTCAACTCGGTCAAAGAG
TCACCCCACGAGCGCATGCATCGCCACATCGAGCTGGCCTGGGCCTTCTCCAC
GGTCATCGGGACGCT
6. Questions about BLAST
6.1 What is a high scoring sequence pair (HSP)?
6.2 What is the Expect value?
6.3 Why is the following statement false: “The query length and query composition
affects the Expect value”.