CIS 667

transcript

CIS 667

BioinformaticsCleveland 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 Historically, creation/maintenance of

biological sequence databases important

• Biology is being transformed from a purely lab-based science to an information science as well

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

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

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

Others…

• Molecular Evolution • Physical chemistry• Statistics and probability• Database design• Algorithm development• Molecular biology lab methods

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

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

Example Amino Acid

C COOH

Alpha Carbon

Amino GroupCarboxy Group

Side Chain

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

Amino AcidsName One-letter code Three-letter code

Alanine A Ala

Cysteine C Cys

Aspartic Acid D Asp

Glutamic Acid E Glu

Phenylalanine F Phe

Glycine G Gly

Histidine H His

Isoleucine I Ile

Lysine K Lys

Leucine L Leu

Methionine M Met

Asparagine N Asn

Proline P Pro

Glutamine Q Gln

Arginine R Arg

Serine S Ser

Threonine T Thr

Valine V Val

Tryptophan W Trp

Tyrosine Y Tyr

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

Proteins

• Proteins fold in three dimensions resulting in secondary, tertiary, quaternary structures

• The two most common secondary structures are the -helix and the -sheet

Secondary Structure

• Only a small number of patterns are common

• Patterns formed by regular intramolecular hydrogen bonding patterns

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

Ribosomes

Large subunit Small subunit

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

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

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)

• 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

DNA Double Helix

• 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

• 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

• 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

• 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)

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

RNA Transcription

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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

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

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

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

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

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)

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

Prokaryotes and Eukaryotes

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

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.

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

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

Restriction Enzyme Digests

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

Gel Electrophoresis

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

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

DNA Chip

Cloning

• Large amounts of DNA material is typically required for analysis In cloning, specific DNA fragments are

inserted into chromosome-like carriers called vectors in living cells

The identical copies of the fragments are called molecular clones and can be stored in libraries for later study

Vectors are derived from bacteria and yeast chromosomes

Cloning

Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR) is an alternative to cloning for amplifying DNA fragments DNA fragments are heated to break

them into single strands Probes are added to bind to the portion

of DNA to be amplified DNA polymerase grows the strands from

the probes The process repeats

Polymerase Chain Reaction

CIS 667

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