Chapter 10 Analytical Biotechnology and the Human Genome Chapter Outline Enzyme tests and biosensors DNA-based tests DNA analysis technologies Human genome and genome-based analytical methods 1
Enzyme-based biological tests Enzyme testing is based on (quantifiable) reactions catalyzed by specific enzymes The test includes an enzyme (usually produced by recombinant microorganism) that converts specific molecule to a product that can be detected (and quantified) Must be sensitive enough to detect minor quantities of specific molecules Glucose oxidase test for diabetes O 2 H 2 O 2 Glucose oxidase specifically takes electrons from glucose and uses them to reduce oxygen to hydrogen peroxide This reaction is coupled to peroxidase-catalyzed reaction that colorimetrically detects hydrogen peroxide Calibration curve is established using constant amount of enzyme and varied concentrations of glucose Glucose oxidase-based test is used to measure levels of glucose in blood and serum 2
Biosensors Biosensors are enzyme- or microorganism-based devices designed to detect specific molecules in a sample Input -> detection/measurement -> output Usually coupled to electronics that help to interprete and quantify a signal Single-use first generation biochip Glucose oxidase is immobilized on a chip Glucose present in a sample binds FAD in the active site of glucose oxidase Electrons drawn from glucose through FAD in the oxidation reaction are taken up by ferrocene (mediator molecule) Reduced ferrocene diffuse to the chip and donate their electrons Resulting electrical current is measured it is proportional to glucose concentration 3
Reusable glucose biosensor Glucose oxidase is immobilized on a membrane covered with gel Only small molecules and oxygen can pass through the membrane Glucose oxidase converts glucose into gluconolactone, reducing oxygen to hydrogen peroxide Hydrogen peroxide easily donates its electrones to an electrode, generating electrical current that can be measured Resulting electrical current is proportional to glucose concentration Microbe-based biosensor Usually based on yeast cells immobilized in a polymer gel and mounted on an oxygen electrode Most frequent application: detection of environmental pollution Yeast cells need organic compounds they can use as food oxygen is consumed if cells can degrade the compounds The measurement of water pollution with organic compounds is based on the amount of oxygen consumed by the immobilized cells The amount of oxygen consumed is proportional to the amount of degradable organic compounds in a sample 4
Immunological pregnancy test Based on antibodies recognizing human chorionic gonadotropin (HCG) protein hormone regulating secretion of pregnancy-specific hormone progesterone HCG can be detected in urine applied on a paper strip with 2 antibodies: HCG-antibody (catcher) and an anti-hcg-antibody (detector bonded to a dye) End of a strip is dipped in urine, HCG binds catcher and is carried through paper capplilaries to firmly immobilized detector Binding of a HCG-catcher complex to the detector reveals its colour A control colour line is revealed independently of the HCG presence Other important antibody-based tests Viral infection tests (HIV, hepatitis B etc) based on virus-specific antibodies Myocardial infraction (heart attack) test based on dying heart cells releasing specific proteins that can be recognized by monoclonal antibodies Point-of-Care tests based on measurement of various types of lipids in the blood 5
DNA analysis can be used in disease testing DNA can be cut with various restriction endonucleases that recognize specific nucleotide sequences a mixture of DNA fragments is generated This mixture can be separated in agarose gel electrophoresis according to fragment size Gel with separated DNA fragments is stained with a dye to visualize DNA Each enzyme will produce unique band pattern Southern blotting: detection of a specific DNA sequence 6
Southern blotting: detection of a specific DNA sequence DNA fingerprinting DNA of 2 individuals usually differs by less than 0.1% This difference is sufficient to distinguish 2 individuals using DNA fingerprinting DNA fingerprinting is based on differences in individuals DNA that affect sites for recognition by restriction endonucleases RFLP: Restriction Fragment Length Polymorphism 7
Polymerase Chain Reaction: DNA amplification Polymerase Chain Reaction (PCR) is based on DNA polymerase that can replicate (copy) DNA. PCR is used to produce many copies of DNA moleucle from a single copy Requires knowledge of DNA nucleotide sequence PCR 8
Gene sequencing Gene sequencing 9
Automatic gene sequencing During sequencing reaction, each DNA product is labeled with a fluorescent dye Sequencing reaction products are separated by capillary electrophoresis At the exis of a capillary, a laser exites the fluorescent dye Camera detects the colour of emitted light, and passes the information to the computer Computer determines DNA sequence from the information on the product size and colour of emitted light FISH (fluorescence in situ hybridization): detection of gene location and copy number 10
Genome & Genomics Genome - the complete copy of the genetic information or one complete set of chromosomes of an organism. Genomics - mapping, sequencing, and analyzing the functions of entire genomes Functional genomics - the study of genome function (transcriptomics + proteomics) Goals of the Human Genome Project (1990-2005) To map all of the human genes To construct a detailed physical map of the entire human genome To determine the nucleotide sequences of all 23 human chromosomes. 11
Human genome: initial analysis Genetic mapping - is based on specific markers that can be associated with particular traits. Distances between markers are estimated from frequency of recombination between them Physical mapping determines positions of specific DNA sequences on the DNA molecule using DNA hybridization and/or restriction endonucleases Human genome: advanced analysis DNA sequencing a shotgun method Bioinformatics development of computer algorithms for assembly and analysis of DNA sequences DNA chips studies of gene expression profiles (transcriptomics) Proteomics identification of all proteins in a cell 12
Pharmacogenomics: toward personalized medical care What you need to know for the exam What is an enzyme-based biosensor and how it works (example) What is an organism-based biosensor and how it works (example) How do immunological tests work (example) How DNA can be analyzed (gel electrophoresis, Southern blotting, PCR, DNA sequencing, FISH) Definition of genome, genomics, functional genomics, genome mapping Initial and advanced genome analyses Explain what is pharmacogenomics 13