BME280CSE277CSE377: Bioinformatics Spring 2006

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BME280/CSE277/CSE377 BioinformaticsSpring 2006
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Administrivia

• Lecture time TTh 1230-145pm
• Lecture place Engineering II, Room 322
• Instructor Ion Mandoiu
• Office ITEB 261
• Tel 6-3784
• E-mail ion_at_engr.uconn.edu
• Office hours MW 1-2pm

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Textbooks

• Neil C. Jones and Pavel A. Pevzner, An
  Introduction to Bioinformatics Algorithms, MIT
  Press, 2004. Textbook website http//bioalgorithm
  s.info/. (REQUIRED)
• D. Gusfield, Algorithms on Strings, Trees, and
  Sequences, Cambridge University Press, 1997
  (OPTIONAL)

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Grading

• 30 homework assignments
• Bi-weekly
• 30 programming projects
• Individual, 3-4 projects
• 40 final project
• Individual or teams of 2
• Written report short presentation
• Possible topics
• Algorithm implementation empirical study
• In-depth survey of a topic not covered in class
• Progress on open research problems
• Propose your own!

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What is Bioinformatics?

• Bioinformatics is generally defined as the
  analysis, prediction, and modeling of biological
  data with the help of computers

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Why Bioinformatics?

• DNA sequencing technologies have created massive
  amounts of information that can only be
  efficiently analyzed with computers
• Hundreds of species sequenced
• Human, rat, chimp, chicken,
• As the information becomes ever so larger and
  more complex, more computational tools are needed
  to sort through the data.
• Biology is becoming an information science!
• Slowly, we are learning how cells work through
  comparative genomics -- not unlike comparative
  linguistics

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Bioinformatics Tools

• Bioinformatics problems involve multiple aspects
• Example Sequence Comparison
• Biology How are genes evolving? How is gene
  function related to gene sequence?
• Learning/AI How do we define similar? Can
  we learn from examples?
• Algorithms How can we efficiently find all
  similar sequences?
• Statistics How do we distinguish a random match
  from a true one?

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Course Description

• Course emphasis
• Modeling computational problems arising in
  biology as graph-theoretic, statistical, or
  mathematical optimization problems
• Design, analysis, and implementation of efficient
  algorithms
• Algorithmic techniques to be covered
• Exhaustive search
• Integer programming
• Greedy algorithms
• Dynamic programming
• Divide-and-conquer
• Graph algorithms
• Combinatorial pattern matching
• Clustering
• Hidden Markov models
• Randomized algorithms

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Course Description

• Biological applications
• Restriction mapping
• DNA sequencing
• Motif finding
• Pairwise sequence alignment
• Gene prediction
• Evolutionary trees
• Genome rearrangements

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Complete and return the survey!
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Basic Molecular Biology
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The Cell
Source D. Geiger
All cells contain the same DNA, yet there are
many types of cells!
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Mendel and his Genes

• Genes -- physical and functional traits passed on
  from one generation to the next
• Discovered by Gregor Mendel in the 1860s while he
  was experimenting with the pea plant. He asked
  the question

Do traits come from a blend of both parent's
traits or from only one parent?
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The Pea Plant Experiments

• Mendel discovered that genes were passed on to
  offspring by both parents in two forms dominant
  and recessive.

• The dominant form would be the phenotypic
  characteristic of the offspring

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DNA The Code of Life

• The structure and the four genomic letters code
  for all living organisms
• Adenine, Guanine, Thymine, and Cytosine which
  pair A-T and C-G on complimentary strands.

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DNA Components
Source D. Geiger
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The Human Genome
Source D. Geiger
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DNA Organization
Source D. Geiger
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Genome Sizes

• E. Coli (bacteria) 4.7 Mb (Mega bases)
• Yeast (simple fungi) 15 Mb
• Nematode (C. Elegans) 100 Mb
• Mouse 2 Gb (Giga bases)
• Human 3 Gb
• Wheat 16.5 Gb
• Lily 32-48 Gb

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Genes

• DNA strings contain
• Coding regions (genes)
• Control regions
• Junk DNA (unknown function)
• Estimated number of genes
• E. Coli (bacteria) 4,000
• Yeast (simple fungi) 6,000
• Nematode (C. Elegans) 13,000
• Human 32,000 (?)

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Central Dogma

• Cells express different subsets of genes under
  different environments

Transcription
Translation
Protein
mRNA
Gene
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Gene Transcription
Source D. Geiger

• RNA similar to DNA, but has
• slightly different backbone
• Uracil (U) instead of Thymine (T)

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RNA Roles
Source D. Geiger
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Translation

• Catalyzed by Ribosome
• Using two different sites, the Ribosome
  continually binds tRNA, joins the amino acids
  together and moves to the next location along the
  mRNA
• 10 codons/second, but multiple translations can
  occur simultaneously

http//wong.scripps.edu/PIX/ribosome.jpg
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Genetic Code

• Human cells produce approx. 100,000 proteins
• Proteins are poly-peptides consisting of
  70-3,000 amino acids
• There are 20 different amino acids every 3
  nucleotides in a gene encode for 1 amino acid (or
  the STOP signal)

Source D. Geiger
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Protein Folding

• Proteins are not linear structures, though they
  are built that way
• The amino acids have very different chemical
  properties they interact with each other after
  the protein is built
• This causes the protein to start fold and
  adopting its functional structure
• Proteins may fold in reaction to some ions, and
  several separate chains of peptides may join
  together through their hydrophobic and
  hydrophilic amino acids to form a polymer

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Protein Folding (contd)

• The structure that a protein adopts is vital to
  its chemistry
• Its structure determines which of its amino acids
  are exposed carry out the proteins function
• Its structure also determines what substrates it
  can react with

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Protein Structure
Source D. Geiger
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Basic Molecular BiotechnologyHow is information
accessed at molecular level?
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Operations on DNA/RNA

• Amplification (making many copies)
• Cutting into shorter fragments
• Reading fragment lengths
• Reading DNA sequence
• Probing presence of specific fragments

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Why we need so many copies

• Biologists needed to find a way to read DNA
  codes.
• How do you read base pairs that are angstroms in
  size?
• It is not possible to directly look at it due to
  DNAs small size.
• Need to use chemical techniques to detect what
  you are looking for.
• To read something so small, you need a lot of it,
  so that you can actually detect the chemistry.
• Need a way to make many copies of the base pairs,
  and a method for reading the pairs.

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Polymerase Chain Reaction

• Problem Modern instrumentation cannot easily
  detect single molecules of DNA, making
  amplification a prerequisite for further analysis
• Solution PCR doubles the number of DNA fragments
  at every iteration

1 2 4 8
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Denaturation
Raise temperature to 94oC to separate the duplex
form of DNA into single strands
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Design primers

• To perform PCR, a 10-20bp sequence on either side
  of the sequence to be amplified must be known
  because DNA pol requires a primer to synthesize a
  new strand of DNA

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Annealing

• Anneal primers at 50-65oC

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Annealing

• Anneal primers at 50-65oC

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Extension

• Extend primers raise temp to 72oC, allowing Taq
  pol to attach at each priming site and extend a
  new DNA strand

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Extension

• Extend primers raise temp to 72oC, allowing Taq
  pol to attach at each priming site and extend a
  new DNA strand

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Repeat

• Repeat the Denature, Anneal, Extension steps at
  their respective temperatures

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Polymerase Chain Reaction
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Restriction Enzymes

• Discovered in the early 1970s
• Used as a defense mechanism by bacteria to break
  down the DNA of attacking viruses.
• They cut the DNA into small fragments.
• Can also be used to cut the DNA of organisms.
• This allows the DNA sequence to be in a more
  manageable bite-size pieces.
• It is then possible using standard purification
  techniques to single out certain fragments and
  duplicate them to macroscopic quantities.

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Molecular Scissors
Molecular Cell Biology, 4th editionfig 9-10
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Discovering Restriction Enzymes

• HindII first restriction enzyme discovered by
  Hamilton Smith in 1970
• From bacterium Haemophilus influenzae
• Discovered accidentally while studying how the
  bacterium Haemophilus influenzae takes up DNA
  from the phage virus P22
• Recognizes and cuts DNA at sequences
• GTGCAC
• GTTAAC

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Recognition Sites of Restriction Enzymes
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Separating DNA by Size

• Gel electrophoresis is a process for separating
  DNA by size
• Can separate DNA fragments that differ in length
  in only 1 nucleotide for fragments up to 500
  nucleotides long

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Gel Electrophoresis

• DNA fragments are injected into a gel positioned
  in an electric field
• DNA are negatively charged near neutral pH
• The ribose phosphate backbone of each nucleotide
  is acidic DNA has an overall negative charge
• DNA molecules move towards the positive electrode

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Gel Electrophoresis (contd)

• DNA fragments of different lengths are separated
  according to size
• Smaller molecules move through the gel matrix
  more readily than larger molecules
• The gel matrix restricts random diffusion so
  molecules of different lengths separate into bands

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Detecting DNA Autoradiography

• One way to visualize separated DNA bands on a gel
  is autoradiography
• The DNA is radioactively labeled
• The gel is laid against a sheet of photographic
  film in the dark, exposing the film at the
  positions where the DNA is present.

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Detecting DNA Fluorescence

• Another way to visualize DNA bands in gel is
  fluorescence
• The gel is incubated with a solution containing
  the fluorescent dye ethidium
• Ethidium binds to the DNA
• The DNA lights up when the gel is exposed to
  ultraviolet light.

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Gel Electrophoresis Example
Direction of DNA movement
Smaller fragments travel farther
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Sequencing

• Biologists can reliably find the sequence of
  A/C/T/G for short strings (few hundred
  nucleotides)
• Chain termination
• Single strand template
• Complementary strand synthesis blocked with small
  probability at particular nucleotides
• Lengths of fragments read for each class of
  strings

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Sequencing

• Biologists can reliably find the sequence of
  A/C/T/G for short strings (few hundred
  nucleotides)
• Chain termination
• Single strand template
• Complementary strand synthesis blocked with small
  probability at particular nucleotides
• Lengths of fragments read for each class of
  strings

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Sequencing

• Biologists can reliably find the sequence of
  A/C/T/G for short strings (few hundred
  nucleotides)
• Chain termination
• Single strand template
• Complementary strand synthesis blocked with small
  probability at particular nucleotides
• Lengths of fragments read for each class of
  strings

ATACGGA ATACGG ATACG ATAC ATA AT A
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Sequencing
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DNA Hybridization

• Single-stranded DNA will naturally bind to
  complementary strands
• Hybridization is used to locate genes, regulate
  gene expression, and determine the degree of
  similarity between DNA from different sources
• Hybridization is also referred to as annealing or
  renaturation

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Microarray Technologies

• Oligonucleotide arrays
• Short (20-60bp) synthetic DNA strands
• Arrays of cDNAs
• Obtained by reverse transcription from Expressed
  Sequence Tags (ESTs)

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DNA Array Hybridization Experiment
Images courtesy of Affymetrix.
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Two-Color Technique

• Sample labeled RED
• Control labeled GREEN
• YELLOW probes hybridize to both sample and
  control
• BLACK probes hybridize to neither

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Sequencing by Hybridization

• Exploits parallel hybridization capabilities
  offered by DNA arrays
• ALL probes of a certain length k (k8 to 10) are
  synthesized on the array
• Target DNA hybridizes at locations which store
  probes complementary to its k-substrings
• Sequencing by Hybridization (SBH) Problem
  Reconstruct target DNA given its k-length
  substrings (spectrum)

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Operations on Proteins

• Cloning in expression vectors
• 2-Dimensional gel electrophoresis separate
  proteins by molecular weight/pH gradient
• Antibody techniques (immunoprecipitation,
  antibody arrays,)
• Mass spectrometry (e.g., MALDI-TOF)

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Active research problems

• Genome projects have already given draft genome
  sequence for hundreds of species, but lots of
  questions remain to be answered
• Create a complete parts list gene sequences
  (including intron/exon structure), transcription
  factors,
• Understand function of each part, e.g., protein
  structure, protein/DNA and protein/protein
  interactions
• Understand mechanisms, e.g., pathways
• Understand how everything fits together systems
  biology