Nucleic acid-based methods (I)

Structure of DNA

Complementality of DNA (high specificity in base paring)

Replication of DNA

Easy manipulation of DNA

Hybridization

Nucleic acid-based methods Gene probing Colony hybridization Southern/Northern hybridization Microarray Polymerase Chain Reaction (PCR) PCR RT-PCR Multiplex PCR Nested PCR

Gene probes Small pieces of DNA complementary to the target sequence of interest Labeling options Radioactive chemicals Nonradioactive alternatives (DIG, biotin, or fluorescein)

Gene probe detection

Colony hybridization

Southern hybridization

Gel electrophoresis

Gel electrophoresis (I)

Gel electrophoresis (II)

DNA on a gel

Southern hybridization

An application of southern hybridization (forensic science)

Microbe Nucleic Acid Detection by DNA Microarrays or “Gene Chip” Technology Generate/obtain DNA complimentary to genes (sequences) of interest; 1000s of different ones Apply tiny quantities of each different one onto solid surfaces at defined positions “gene chip” or “DNA microarray” Isolate or amplify target NA of interest and label with a fluorescent probe Apply sample NA to the “gene chip” surface Sample NA binds to specific DNA probes on chip surface; wash away unbound NA Detect bound DNA or RNA by fluorescence after laser excitation Analyze hybridization data using imaging systems and computer software Fluorescing Gene Chip or DNA Microarray

Polymerase Chain Reaction (PCR)

Primers and enzymes Primers Taq polymerase Short nucleotides complementary to a target DNA Upstream and downstream primers Taq polymerase Heat stable DNA-dependant DNA polymerase Isolated from thermophilic bacteria (Thermus aquaticus) Withstand temperature up to 98oC

Different PCR techniques RT-PCR Multiplex PCR Nested PCR

Agarose Gel Electrophoresis Separate nucleic acid fragments in an agarose gel Resolves small DNA molecules: 0.1 to 50 kb % agarose determines resolution of DNA size: 0.3% w/v: resolves 5 to 50 kb 2% w/v resolves 0.1 to 2 kb Resolving large molecules (up to 500 kb) requires specialized methods Pulse-field gel electrophoresis (PFGE) DNA marker ladder Specific DNA fragment

Example: RT-PCR and Oligoprobe Detection of Enteroviruses in Water

Real-Time PCR and Quantitative Fluorogenic Detection Molecular beacon. Several 5' bases form base pairs with several 3' bases; reporter and quencher in close proximity. If reporter is excited by light, its emission is absorbed by quencher & no fluorescence is detected. Detection of PCR product by molecular beacon. Beacon binds to PCR product and fluoresces when excited by the appropriate  of light. [Fluorescence] proportional to [PCR product amplified]