M.Sc. II BiotechnologyAnimal Biotechnology

Dariyus Z Kabraji

Bioinformatics in Mutation Detection

M.Sc. II BiotechnologyAnimal Biotechnology

Dariyus Z Kabraji

Introduction• Mutation is a permanent change of the nucleotide

sequence of the genome of an organism, virus, or extra-chromosomal DNA or other genetic elements.

• Result from damage to DNA which is not repaired or to RNA genomes (typically caused by radiation or chemical mutagens), errors in the process of replication, or from the insertion or deletion of segments of DNA by mobile genetic elements.

• Can either have no effect, alter the product of a gene, or prevent the gene from functioning properly or completely.

Sequence Analysis• Sequencing can be used to find genes, segments of

DNA that code for a specific protein or phenotype• If a region of DNA has been sequenced, it can be

screened for characteristic features of genes.• Way of arranging the sequences of DNA, RNA or

protein to identify regions of similarity .• Helps in inferring functional , Structural or

evolutionary relationship between the sequence• Used to find the best- matching sequences

Alignment - the Key• Alignment is the task of locating “equivalent”

regions of two or more sequences to maximize their similarity

WILLIAM SHAKESPEARE WILLIAM SHAGSPUR— Red indicates mismatches and white indicates gaps

Principles of Sequence Alignment

• Alignment can reveal homology between sequences• Similarity is descriptive term that tells about the

degree of match between the two sequences• Sequence similarity does not always imply a

common function• Conserved function does not always imply

similarity at the sequence level• Convergent evolution: sequences are highly similar,

but are not homologous

Filaggrin Gene Mutation• Filaggrins are filament-associated proteins which bind

to keratin fibers in epithelial cells• Normally found in large quantities in the outermost

layers of the skin• Is essential for skin barrier function, helping to form a

protective layer at the surface of the skin• Individuals with truncation mutation in the gene

coding for filaggrin are strongly predisposed to a severe form of dry skin (ichthyosis vulgaris), asthma, and eczema

Filaggrin Gene Mutation• Filaggrins are filament-associated proteins which bind

to keratin fibers in epithelial cells• Normally found in large quantities in the outermost

layers of the skin• Is essential for skin barrier function, helping to form a

protective layer at the surface of the skin• Individuals with truncation mutation in the gene

coding for filaggrin are strongly predisposed to a severe form of dry skin (ichthyosis vulgaris), asthma, and eczema

Objective of Sequence Analysis in this case

• To detect the faulty filaggrin gene which may cause Eczema & Asthma

• To find out the identical human proteins which may have functional similarity with filaggrin

• To find out same or identical proteins from other species which may be helpful in therapeutics

Reading Frames (RF)• A reading frame is a way of dividing the

sequence of nucleotides in a nucleic acid (DNA or RNA) molecule into a set of consecutive, non-overlapping triplets.

• Where these triplets equate to amino acids or stop signals during translation, they are called codons.

Reading Frames (RF)AGG·TGA·CAC·CGC·AAG·CCT·TAT·ATT·AGCA·GGT·GAC·ACC·GCA·AGC·CTT·ATA·TTA·GCAG·GTG·ACA·CCG·CAA·GCC·TTA·TAT·TAG·C

Open Reading Frame (ORF)• An open reading frame (ORF) is the part of a reading frame that has the

potential to code for a protein or peptide. • An ORF is a continuous stretch of codons beginning with a start codon (usually

ATG) and ending with a stop codon (usually TAA, TAG or TGA). One common use of open reading frames is as one piece of evidence to assist in gene prediction.

• Long ORFs are often used, along with other evidence, to initially identify candidate protein coding regions in a DNA sequence.

Three tools for determining ORF:1. Finder2. Investigator3. Predictor

ORFPredictor• OrfPredictor is a web server designed for identifying protein-

coding regions in expressed sequence tag (EST)-derived sequences.

• For query sequences with a hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments; otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences.

• The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where the coding region begins and ends.

• OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects.

Sequence Retrieval• The simplest input for RSAT is a list of gene names. • Using this list the retrieve-seq program returns upstream,

downstream or unspliced ORF sequences (introns and spliced ORFs will soon be supported).

• The user can specify the left and right limits of the sequences to be retrieved.

• Default values have been selected for each genome, depending on the average size of the intergenic regions and mechanisms of regulation.

• Upstream sequences can be retrieved over a constant size, but an option also allows to clip them in order to avoid the inclusion of coding sequences from upstream ORFs.

Anticipated Prospects• Preventive measures for susceptible people• Therapeutic -Identify similar proteins which might

have similar functions as that of filaggrin.• Plant & Animal proteins – Proteins identical with

filaggrin in plants and animals can be utilized for protein supplements

• Drugs or other treatments aimed at the filaggrin gene are still some years away but research in this direction give hope to those with these distressing conditions.

References• Filaggrin mutations and the skin; Dipankar De,

Sanjeev Handa; Contact Dermatitis; Year : 2012, Volume : 78. Issue : 5, Page : 545-551

• Regulatory Sequence Analysis Tools; Jacques van Helden; Nucleic Acids Research, 2003, Vol. 31, No. 13 3593–3596 DOI: 10.1093/nar/gkg567