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CIS 667

CIS 667. Bioinformatics Cleveland State University Department of Computer and Information Science Fall 2003. What is Bioinformatics?. Field of science in which biology, computer science, information technology merge to form a single discipline

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CIS 667

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  1. CIS 667 Bioinformatics Cleveland State University Department of Computer and Information Science Fall 2003

  2. What is Bioinformatics? • Field of science in which biology, computer science, information technology merge to form a single discipline • Historically, creation/maintenance of biological sequence databases important • Biology is being transformed from a purely lab-based science to an information science as well

  3. What is Bioinformatics? • Three important sub-disciplines • Development of new algorithms and statistical methods to analyze relationships among members of large data sets • Analysis and interpretation of various types of data (nucleotide and amino acid sequences, protein structures, etc.) • Development/implementation of tools for efficient access/mgmt. of various types data

  4. Why now? • Recent advances in molecular biology and genomic technologies lead to an explosive growth in the amount of biological information generated • Requires computerized databases to store/organize/index data and specialized tools to view and analyze data

  5. What skills should a Bioinformatician have? • Deep background in some area of molecular biology • Understand the central dogma of molecular biology • Substantial experience with at least one or two major packages • Experience working in a command-line computing environment • Experience with both high-level and scripting languages

  6. Others… • Molecular Evolution • Physical chemistry • Statistics and probability • Database design • Algorithm development • Molecular biology lab methods

  7. What will we learn? • Central dogma of molecular biology + other necessary biology background • Working in a Unix command-line environment • Programming in Perl • Algorithms for molecular biology • Hands-on experience with bioinformatics tools

  8. Molecular Biology • Primarily concerned with two basic molecules of all living things: • Proteins • Structural proteins are tissue building blocks while enzymes catalyze chemical reactions • Proteins are chains of amino acids

  9. Side Chain Alpha Carbon Carboxy Group Amino Group Example Amino Acid CH3 C H2N COOH H

  10. Amino Acids • There are 20 naturally occurring amino acids • Amino acids can be identified by a 3-letter code (and sometimes by 1-letter code) • In a protein, amino acids are joined by peptide bonds (C from carboxy group binds to N from amino group) • A water molecule is liberated so we speak of residues in the chain

  11. Amino Acids

  12. Proteins • Typical protein contains about 300 residues • Chain have an amino group at one end and a carboxy group at the other giving the chain an orientation (start - end) • The sequence of residues in the chain is called the protein’s primary structure

  13. Proteins • Proteins fold in three dimensions resulting in secondary, tertiary, quaternary structures • The two most common secondary structures are the-helix and the -sheet

  14. Secondary Structure • Only a small number of patterns are common • Patterns formed by regular intramolecular hydrogen bonding patterns

  15. Proteins • The specific shape that a protein folds into determines its unique function • Different shapes mean the protein can bind to different molecules • Proteins are produced in a cell structure called a ribosome • Amino acids are added one after the other in the sequence coded by a messenger ribonucleic acid (mRNA) molecule

  16. Ribosomes Large subunit Small subunit

  17. Nucleic Acid • Two types of nucleic acids • Ribonucleic acid (RNA) • Deoxyribonucleic acid (DNA) • DNA, like protein, is a chain of simpler molecules, but double stranded • Each strand consists of a chain of nucleotides

  18. Nucleic Acids • Each nucleotide consists of • A sugar molecule • A phosphate residue • A base • The sugar molecule has five carbon atoms labeled 1’ - 5’ • The 3’ carbon of one nucleotide is bound to the 5’ carbon of the next nucleotide in the chain giving an orientation to the chain • 5’ is the start and 3’ is the end

  19. Nucleic Acids

  20. Nucleic Acids • The chain of sugar/phosphate groups forms the backbone of a strand of DNA • Attached to each 1’ carbon in the backbone is a molecule called a base • There are four different bases • Adenine (A) • Guanine (G) • Cytosine (C) • Thymine (T)

  21. DNA • DNA molecules are double strands • The strands form a double helix • The strands are held in the helix form by bonds between complementary bases in the two strands • A and T are complements • G and C are complements • We refer to the paired bases as base pairs (bp) and use base pairs as the unit of length of DNA molecules

  22. DNA Double Helix

  23. DNA • DNA can be considered as a string of letters from the set {A, T, C, G} • 5’ … TACTGAA … 3’ • This other strand connected to this one is antiparallel and complentary • 3’ … ATGACTT … 5’ • Note that the orientations of the two strands are opposite

  24. DNA • Given one of the strands, we can infer the other strands • One of the strands can act as a template for the construction of the other • This property allows for cell division and replication with each new cell containing a copy of the DNA from the original cell • Complementary base pairs are held together by hydrogen bonds

  25. DNA • In higher organisms, DNA is found inside the cell nucleus • Also in cell organelles called mitochondria (plants and animals) and chloroplasts (plants only) • The DNA is found in a few very long DNA molecules called chromosomes

  26. RNA • RNA molecules are similar to DNA, but • Have a different sugar • Have the base uracil (U) instead of thymine (T) • U binds with A, as does T • RNA does not form a double helix • Hybrid DNA-RNA helices may form • Parts of an RNA molecule may bind to other parts of the same molecule by complementarity • Three-dimensional structure is variable (compare Protein)

  27. Central Dogma of Molecular Biology • Information stored in DNA is used to make a transient RNA • Process is called transcription accomplished through use of enzyme RNA polymerase • The RNA is used to make proteins • Process is called translation and is performed by ribosomes

  28. RNA Transcription

  29. RNA Transcription

  30. Genes and the Genetic Code • All of the proteins in an organism are specified by a contiguous stretch of DNA called a gene • Remember that the DNA is contained in a small number of molecules called chromosomes • Not all of the DNA specifies some protein • Some genes code for RNA products

  31. Gene Expression • Gene expression is the process of using the information stored in DNA to make an RNA molecule and then a protein • RNA polymerases must • determine the start of genes • determine whether the protein coded by a gene is needed at the present moment • Start of gene marked by 13 nucleotides (why 13, not, e.g. 1) promoter sequence

  32. Gene Expression

  33. Gene Expression • How does the RNA polymerase then tell if a protein should now be produced? • Specific regulatory genes produce proteins capable of binding to a cell’s DNA near the promoter sequence of a gene they control in some circumstances • Positive regulation when binding makes RNA polymerase initiation of transcription easier, negative regulation when harder

  34. Genetic Code • A gene codes the sequence of amino acids needed to form a protein • 20 aa > 4 bases  need more than one base to specify an aa • 43 > 20, so 3 bases suffice • Each sequence of 3 bases (a codon) codes for an amino acid (with 3 exceptions) • Three codons cause translation to end and are called stop codons

  35. Genetic Code • Since 64 > 20, more than one codon must code for some amino acid(s) • In fact, 18 of the 20 amino acids are coded for by more than one codon • The genetic code is therefore a degenerate code • Errors in transcription may not cause the wrong aa to be produced (especially if the error is in the 3rd nucleotide) • Even if the wrong aa is produced due to a single error, a similar aa is likely to be produced

  36. Open Reading Frames • One special start codon (AUG) marks the spot where translation begins • A sequence of codons is called a reading frame • A sequence of codons which begins with a start codon and has no stop codons is called an open reading frame (orf)

  37. Prokaryotes and Eukaryotes • Living organisms may be classified as either prokaryote (bacteria) or eukaryote (higer organisms like yeast, plants, people) • The cells of eukaryotes have a nucleus while prokaryotes don’t • DNA is linear in eukaryotes and circular in prokaryotes

  38. Prokaryotes and Eukaryotes

  39. Introns and Exons • In prokaryotes, the mRNA copies of the genes corresponds directly to the DNA sequence in the genome (with U substituted for T) • In eukaryotes, the mRNA is carried outside the nucleus before translation • The mRNA is modified by splicing out sequences of introns and rejoining the exons that flank them

  40. Introns and Exons • Splicing is controlled by enzyme complexes called spliceosomes • Incorrect splicing leads to frame shifts or premature stop codons which make the resulting protein useless • The position of introns is signalled by several specific sequences of nucleotides • Since there is more than one sequence we can have alternative splicing resulting in different proteins being produced in different circumstances.

  41. Molecular Biology Tools • A small set of laboratory techniques are used by molecular biologists to identify the information content of organisms so that it can be processed using bioinformatics methods

  42. Restriction Enzyme Digests • Restriction enzymes can be used to cut DNA molecules wherever a particular sequence occurs • Digesting a DNA molecule and observing how many fragments occur gives some insight into the organization and sequence of that DNA • This is called restriction mapping • Allows isolation and experimentation of individual genes for the first time

  43. Restriction Enzyme Digests

  44. Gel Electrophoresis • We can separate the fragments of DNA obtained by restriction enzymes with gel electrophoresis • DNA fragments are pulled through a gel towards an electrical charge • Larger fragments do not move as quickly, so this provides a way to separate the fragments by size

  45. Gel Electrophoresis

  46. Blotting and Hybridization • To study a single fragment, DNA is transferred from the gel to a piece of paper or cloth (blotting) • The DNA fragments are then permanently attached to the membrane using (e.g.) UV light • A specially prepared labeled fragment of DNA (a probe) is allowed to base pair with the fragments to try to find a specific fragment

  47. Blotting and Hybridization • The probe is tagged using (e.g.) a fluorescent dye (hybridization) • Then determine where on the membrane base pairing has occurred • DNA chip or microarray techniques are similar • Thousands of nucleotide sequences are affixed to portions of a small silica chip • A large number of probes are washed over the chip and a laser is used to find which probes bind to which sequences

  48. DNA Chip

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