Sequence Alignments—part 2 Week 4

Independent Project Gene Due 9/25 (Monday) must be submitted by email Quiz 1: 10/9 ~35 questions theory and practical (questions based on the programs and websites we have used). Paper quiz, no computer work.

Review for quiz

Review for quiz

Review for quiz

Review for quiz

Review for quiz

Review for quiz

Learning Objectives: Learn to use Online Mendelian Inheritance in Man (OMIM) Understand the different types of mutations and how they can alter protein products Use EMBOSS-Needle to generate a pairwise alignment Determine if mutations fall within coding sequences or in untranslated regions (UTR)

Identifying mutations in genes of interest DNA RNA protein phenotype Bioinformatics and Functional Genomics, 2nd Edition. http://www.bioinfbook.org (2014).

A genetic map of Human Chromosome 7 Location of the gene responsible for Cystic Fibrosis in 90% of people of Northern European descent

Sequence of the Cystic Fibrosis Gene: CFTR

The deletion of these three base pairs is responsible

DNA uses a triplet code to specify amino acids DNA sequence: GGGAGTGTAGATCGT GGG AGT GTA GAT CGT Gly—Ser—Val—Asp—Arg Codons: Amino acids: 15

Mutation resulting in a single base pair substitution GGG AGT GTA GAT CGT GGG AGT GCA GAT CGT Original Sequence Base substitution Result is an amino acid substitution Val>Ala 16

GTA codes for Val (V) while GCA codes for Ala (A) Korf, Y., Yandell, M. & Bedell, J. BLAST: an essential Guide to the Basic Local Alignment Search Tool (Sebastopol; O’Reilly, 2003).

Single Base Substitutions Neutral: No amino acid change Missense: Amino acid substitution (change) Nonsense: Changes codon to a termination sequence (leads to a truncated protein) (stop) 18

An example of a neutral change: GTA>GTG Korf, Y., Yandell, M. & Bedell, J. BLAST: an essential Guide to the Basic Local Alignment Search Tool (Sebastopol; O’Reilly, 2003).

Single Base Substitutions Neutral: No amino acid change Missense: Amino acid substitution (change) Nonsense: Changes codon to a termination sequence (leads to a truncated protein) (stop) 20

GTA codes for Val (V) while GCA codes for Ala (A) http://www.chemguide.co.uk/organicprops/aminoacids/dnacode.gif

Single Base Substitutions Neutral: No amino acid change Missense: Amino acid substitution (change) Nonsense: Changes codon to a termination sequence (leads to a truncated protein) (stop) 22

Mutations that results in a TER/stop codon Korf, Y., Yandell, M. & Bedell, J. BLAST: an essential Guide to the Basic Local Alignment Search Tool (Sebastopol; O’Reilly, 2003).

Mutation resulting in a single base pair deletion Original Sequence GGG AGT GTA GAT CGT GGG AGT GAG ATC GT Base Deletion Result is an alteration in many codons 24

Mutation resulting in a single base pair deletion Original Sequence GGG AGT GTA GAT CGT Gly—Ser—Val—Asp—Arg Gly—Ser—Glu—Ile— ? GGG AGT GAG ATC GT? Base Deletion Result is an alteration in many codons 25

Mutation resulting in a single base pair insertion Original Sequence GGG AGT GTA GAT CGT GGG AGT GCT AGA TCG T Base Deletion Result is an alteration in many codons 26

Mutation resulting in a single base pair insertion Original Sequence GGG AGT GTA GAT CGT GGG AGT GCT AGA TCG T Gly—Ser—Val—Asp—Arg Gly—Ser—Ala—Arg—Ser Base Deletion Result is an alteration in many codons 27

Insertions and Deletions are often referred to as InDels GGG AGT GTA GAT CGT GGG AGT GTT AGA TCG T Sequence 1 Sequence 2 28

Insertions and Deletions are often referred to as InDels GGG AGT GTA GAT CGT GGG AGT GTT AGA TCG T Sequence 1 Sequence 2 Is it an insertion in sequence 2 or a deletion in sequence 1? 29

Insertions and deletions can affect large regions of sequence Entire chromosomes can be deleted (lost) 30

Duplications are a specialized type of insertion Genes Repeats Location 31

Trinucleotide Repeats are examples of duplications ~30 genetic diseases in humans are due to expansion of simple repeats. Genes Repeats Location 32

Where to find OMIM?

How to use OMIM

How to use OMIM

OMIM links genotype to phenotype

OMIM catalogues disorders/diseases

Allelic variants describe mutations in genes

Table view gives a summary of variants

How to use OMIM

How to use OMIM GLY12SER: Glycine at position12 substituted with Serine 3-BP INS, GLY11INS: 3-base pair insertion, Glycine inserted at position 11

How to use OMIM GLY12SER: Glycine at position12 substituted with Serine 3-BP INS, GLY11INS: 3-base pair insertion, Glycine inserted at position 11

How to use OMIM

Common abbreviations in OMIM DEL: Deletion INS: Insertion DUP: Duplication TER: Terminator/Stop Codon Amino acids are abbreviated using 3 letter code: PRO: Proline Nucleotides are abbreviated using 1 letter code: A,G,T and C NT: Nucleotide (usually referring to position i.e. NT305) BP: Base pair (3BP INS: three base pair insertion) KB: Kilo Base (1000 Bases) IVS: Intervening sequence (intron) (sometimes noted as +1 or -1)

Generate a pairwise sequence comparison http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html

Paste FASTA formatted text files

Pairwise alignment allows you to identify mutations

Pairwise alignment allows you to identify mutations 2 BP DEL at NT position 44 Substitution G to A at NT position 152

Exons are numbered by their location Lodish, H. et al. Molecular Cell Biology (New York; W.H. Freeman, 2000). 52

Exons include 5’UTR, coding sequence and 3’UTR Exon 1 includes 5’UTR and coding sequence Exon 3 includes 3’UTR and coding sequence Lodish, H. et al. Molecular Cell Biology (New York; W.H. Freeman, 2000). 53

Worksheet