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International Livestock Research Institute, Nairobi, Kenya. Introduction to Bioinformatics: NOV. 2005 David Lynn (M.Sc., Ph.D.) Trinity College Dublin Ireland. http://www.binf.org/ILRI2005/. Topics for the next 4 days:. Day 1 – Nucleic Acid Sequence Analysis

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slide1

International Livestock Research Institute,

Nairobi,

Kenya.

Introduction to Bioinformatics:

NOV. 2005

David Lynn (M.Sc., Ph.D.)

Trinity College Dublin

Ireland.

http://www.binf.org/ILRI2005/

topics for the next 4 days
Topics for the next 4 days:
  • Day 1 – Nucleic Acid Sequence Analysis
  • Day 2 – Protein Sequence Analysis
  • Day 3 – Accessing Complete Genomes
  • Day 4 – Alignments & Homology Searching
  • Day 4 – Phylogenetic Trees
day 1
Day 1
  • Introduction
  • Interrogating Sequence Databases
  • Translating DNA in 6 frames.
  • Reverse complement & other tools.
  • Calculating some properties of DNA/RNA sequences.
  • Primer design.
  • Gene prediction.
  • Alternative splicing.
  • Promoter characterisation.
  • Other resources.
1 translating dna in 6 frames
5'3' Frame 1

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I T W Y S I

5'3' Frame 2

atcacctggtatagtataa

S P G I V *

5'3' Frame 3

atcacctggtatagtataa

H L V * Y

3'5' Frame 1

ttatactataccaggtgat

L Y Y T R *

3'5' Frame 2

ttatactataccaggtgat

Y T I P G D

3'5' Frame 3

ttatactataccaggtgat

I L Y Q V

1) Translating DNA in 6 frames
slide5
Why?
  • Translating in all 6 frames is commonly done for a range of bioinformatics applications.
  • One place you may need to do it is to locate ORFs in an mRNA sequence which will have untranslated 3’ and 5’ UTRs.
  • Try find the protein sequence encoded by the IL-11 mRNA (link on webpage) using the Translate Tool at Expasy.
2 search launcher at baylor college
2) Search launcher at Baylor College
  • Readseq – converts sequences from one format to another.
  • RepeatMasker – masks sequences against repeat sequences.
  • Primer Selection - PCR primer selection (See primer design later).
  • WebCutter- restriction maps using enzymes w/ sites >= 6 bases.
  • 6 Frame Translation - translates a nucleic acid sequence in 6 frames.
  • Reverse Complement - reverse complements a nucleic acid sequence.
  • Reverse Sequence - reverses sequence order.
  • Sequence Chopover - cut a large protein/DNA sequence into smaller ones with certain amounts of overlap.
  • HBR - Finds E.coli contamination in human sequences.
3 oligo calculator
3) Oligo Calculator
  • Calculates the
    • Length
    • %GC content
    • Melting temperature (Tm) the midpoint of the temperature range at which the nucleic acid strands separate
    • Molecular weight
    • What an OD = 1 is in picoMolar of your input sequence.
  • Many of these parameters are useful in primer design
beer lambert law
Beer – Lambert Law
  • A = ecl
    • e = molar extinction coefficient
    • c = molar concentration
    • l = light path = 1 cm
  • A = O.D.
  • If O.D. = 1 = 41 pM
  • Reading of O.D. = 0.5 on spectrometer
    • => concentration = 20.5pM
5 gene prediction
5) Gene Prediction
  • Gene prediction is an area under intensive research in bioinformatics.
  • GENSCAN program - one of the major programs used to predict genes in the human genome .
  • Should be useful in predicting genes in most vertebrate species, although caution should be used when dealing with other species especially prokaryotes where other programs are more suitable.
  • The Institute for Genomic Research
  • The Deambulum Nucleic Acids Sequence Analysis page at Infobiogen
6 splice site prediction alternative splicing
6) Splice site prediction/Alternative splicing
  • For proper splicing => some way to distinguish exons from introns.
  • Accomplished using certain base sequences as signals.
  • Allow the spliceosome (the cellular machinery that does the splicing) to identify the 5' and 3' ends of the intron.
  • Eukaryotes: the base sequence of an intron begins with 5' GU, and ends with 3' AG.
  • Each species has additional bases associated with these splice sites.
  • Introns also have another important sequence signal called a branch site containing a tract of pyrimidine bases and a special adenine base, usually approximately 50 bases upstream from the 3' splice site.
alternative splicing
Alternative splicing
  • Central dogma of molecular biology was that 1 gene = 1 protein.
  • Multiple possible mRNA transcripts can be produced from 1 gene and if translated these transcripts can code for very different proteins
    • Alternative splicing
  • 4 basic methods of alternative splicing.
the human alternative splicing database at ucla
The Human Alternative Splicing Database at UCLA
  • Used ESTs to locate alternative splices.
  • Project has resulted in a publication of over six thousand alternatively spliced isoforms of human genes.
  • Search the database using any of the following identifiers:
    • Gene Symbol
    • UniGene Sequence Identifier
    • UniGene Cluster Identifier
    • Gene Title
    • GenBank Sequence Identifier
7 promoter analysis recognition
7) Promoter Analysis & Recognition
  • A promoter is a sequence that is used to initiate and regulate transcription of a gene.
  • Most protein-coding genes in higher eukaryotes have polymerase II dependent promoters.
  • Features of pol II promoters:
    • Combination of multiple individual regulatory elements.
    • Most important elements are transcription factor binding sites.
    • CAAT or TATA boxes are neither necessary nor sufficient for promoter function.
    • In many cases, order and distances of elements are crucial for their function.
    • Sequences between elements within a promoter are usually not conserved and of no known function.
promoterinspector
PromoterInspector
  • predicts eukaryotic pol II promoter regions with high specificity (~ 85%) in mammalian genomic sequences.
  • sensitivity of PromoterInspector is about 50% which means that the current version predicts about every second promoter in the genome.
  • PromoterInspector predicts the approximate location of a promoter region and not the exact location of the Transcription Start Site (TSS).
matinspector professional
MatInspector professional
  • Individual Transcription Factor sites build the basis of the promoter.
  • Relatively short stretches of DNA (10 - 20 nucleotides)
  • Sufficiently conserved in sequence to allow specific recognition by the corresponding transcription factor.
  • Utilizes a library of matrix descriptions for transcription factor binding sites to locate matches in sequences.