1. 9/21/2010 1 Course Outline Molecular diagnostics. Introduction, the use of ELISA, DNA hybridization, PCR,, RAPD and DNA fingerprinting, bacterial biosensors, PCR/OLA, and restriction digest, for DNA diagnostics. Therapeutic agents and vaccines. Enzymes as therapeutic agents, production of monoclonal antibodies in E. coli, nucleic acids as therapeutic agents, HIV therapeutic agents, subunit vaccines, attenuated vaccines, and vector vaccines. The synthesis of commercial products by recombinant micro- organisms. Production of restriction enzymes, ascorbic acid, microbial synthesis of indigo and the production of xanthan gum. *Text: Glick, B.R. and Pasternak, J.J. Molecular Biotechnology, Principles and Applications of Recombinant DNA Technology. Molecular Diagnostics The success of modern medicine depends on the detection of specific molecules e.g.  Viruses  Bacteria  Fungi  Parasites  Proteins In water, plants, soil and humans.

2. 2 9/21/2010 Characteristics of a Detection System A good detection system should have 3 qualities :  Sensitivity  Specificity  Simplicity  Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules.  Specificity: the test yields a positive result for the target molecule only.  Simplicity: the test must be able to run efficiently and inexpensively on a routine basis. Immunological Diagnostic Procedures Immunological diagnostic procedures are often used to: Test drugs  Monitor cancers  Detect pathogens ELISA (Enzyme Linked Immunosorbent Assay) This involves the reaction of an antibody with an antigen and a detection system to determine if a reaction has occurred. ELISA involves : Binding of the test molecule or organism to a solid support e.g. micro titer plate.

3. 3 9/21/2010 ELISA Addition of a specific antibody (primary antibody) which will bind to the test molecule if it is present. Washing to remove unbound molecules. Addition of secondary antibody which will bind to the primary antibody. The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. Wash to remove unbound antibody. Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result.

4. 9/21/2010 4 Detection of HIV DNA Diagnostic Systems DNA Diagnostic Systems include :  DNA Hybridization  PCR  Restriction endonuclease analysis  RAPD (random amplified polymorphic DNA)  DNA fingerprinting

5. 9/21/2010 DNA Hybridization Bacterial and viral pathogens may be pathogenic because of the presence of specific genes or sets of genes. Genetic diseases often are due to mutations or absence of particular gene or genes. These genes (DNA) can be used as diagnostic tools. This involves using a DNA probe during DNA hybridization. DNA Hybridization For DNA hybridization:  A probe is needed which will anneal to the target nucleic acid.  Attach the target to a solid matrix e.g. membrane.  Denaturation of both the probe and target.  Add the denatured probe in a solution to the target.  If there is sequence homology between the target and the probe, the probe will hybridize or anneal to the target.  Detection of the hybridized probe e.g. by autoradiography, chemiluminsence or colorimetric. 5

6. 6 9/21/2010 Detection of Malaria Malaria is caused by the parasite Plasmodium falciparum. What kind of organism is P. falciparum? The parasite infects and destroys red blood cells. Symptoms include fever, rashes and damage to brain, kidney and other organs. Current treatment involves microscopic observations of blood smears, which is labour intensive. Other methods e.g ELISA does not differentiate between past and present infection.

7. 7 9/21/2010 Detection of Malaria A DNA diagnostic system would only measure current infection. The procedure involves : A genomic library of the parasite was screened with probes for parasitic DNA. The probes which hybridized strongly were tested further. The probes were tested for their ability to hybridize to other Plasmodium species which do not cause malaria and to human DNA. Detection of Malaria Probes which hybridized to P. falciparum only could be used as a diagnostic tool. The probe was able to detect 10 pg of purified DNA or 1 ng of DNA in blood smear. Other DNA probes were developed for the following diseases: Salmonella typhi (food poisoning) E. coli (gastroenteritis) Trypanosoma cruzi (chagas’ disease)

8. 8 9/21/2010 Polymerase Chain Reaction PCR uses 2 sequence specific oligionucleotide primers to amplify the target DNA. The presence of the appropriate amplified size fragment confirms the presence of the target. Specific primers are now available for the detection of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi. Using PCR to Detect for HIV RT--PCR (reverse transcriptase PCR). HIV has a ssRNA genome. Lyse plasma cells from the potentially infected person to release HIV RNA genome. The RNA is precipitated using isoproponal. Reverse transciptase is used to make a cDNA copy of the RNA of the virus. This cDNA is used as a template to make dsDNA.

9. 9 9/21/2010 RT--PCR Diagnosis of HIV Using PCR to Detect for HIV Specific primers are used to amplify a 156 bp portion of the HIV gag gene. Using standards the amount of PCR product can be used to determine the viral load. PCR can also be used as a prognostic tool to determine viral load. This method can also be used to determine the effectiveness antiviral therapy. (Brock Biology of Microorganisms 9th ed. pg 883--886).

10. 10 9/21/2010 Restriction Digest Analysis Diagnosis of sickle cell anemia. Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6th aa of the  chain of hemoglobin. A (normal) glutamic acid and S (sickle) valine. In the homozygous state SS the red blood cells are irregularly shaped. The disease results in progressive anemia and damage to heart, lung, brain, joints and other organ systems. This occurs because the mutant hemoglobin is unable to carry enough oxygen to supply these systems. Diagnosis of Sickle Cell Anemia The single mutation in hemoglobin cause a change in the restriction pattern of the  globin gene abolishing a CvnI site. CvnI site CC  TNAGG (N = any nt) Normal DNA sequence CCTGAGG (A) Mutant DNA sequence CCTGTGG (S) Two primers which flank the mutant region of the  globin gene is used during PCR to amplify this region of the gene. The PCR products is digested with CvnI and separated by agarose gel electrophoresis.

11. 11 9/21/2010 Detection of Sickle cell anemia by PCR PCR/OLA Like sickle cell anemia many genetic diseases are caused by mutant genes. Many diseases are caused by a single nucleotide (nt) change in the wild type gene. A single nt change can be detected by PCR/OLA ( oligonucleotide ligation assay). E.g. The normal gene has A at nt position 106 and mutant has a G. 2 short oligonucleotides (oligo) are synthesized Oligo 1 (probe x) is complementary to the wild type has A at 106 (3’ end).

12. 12 9/21/2010 PCR/OLA Oligo 2 (probe y) has G at 107 (5’ end). The two probes are incubated with the PCR amplified target DNA. For the wild type the two probes anneal so that the 3’end of probe x is next to the 5’end of probe y. For the mutant gene the nt at the 3’ end of probe x is a mismatch and does not anneal. PCR/OLA

13. 13 9/21/2010 PCR/OLA DNA ligase is added. The two probes will only ligate if the two probes are perfectly aligned (as in the wild type). To determine if the mutant or wild type gene is present it is necessary to detect for ligation. Probe x is labeled at 5’ end with biotin Probe y is labeled at 5’ end with digoxygenin. PCR/OLA Digoxygenin serves as an antibody binding indicator. After washing a colourless substrate is added. If a coloured substrate appears this is indicative that the biotin probe (x) ligated to the dioxygenin probe (y) and that the wild type gene is present.

14. 14 9/21/2010 PCR/ OLA DNA Fingerprinting (RFLP) RFLP = Restriction Fragment Length Polymorphism Regular fingerprinting analyses phenotypic traits. DNA fingerprinting analyses genotypic traits. DNA fingerprinting (DNA typing) is used to characterize biological samples. In legal proceedings to identify suspects and clear others. Paternity testing

15. 15 9/21/2010 DNA Fingerprinting (RFLP) The procedure involves :  Collection of sample e.g. hair, blood, semen, and skin.  Examination of sample to determine if there is enough DNA for the test.  The DNA is digested with restriction enzymes.  Digested DNA is separated by agarose gel electrophoresis.  DNA is transferred by Southern blotting to a membrane.  Membrane is hybridized with 4--5 different probe  Detection of hybridization. Microsatellite DNA After hybridization the membranes are stripped and reprobed. The probes used are human microsatellite DNA. These sequences occur in the human genome as repeated sequences. E.g ATTAG….ATTAG….ATTAG…. The length of the repeat is 99--40 bases occurring 10--30 times. The microsatellites have different length and numbers in different individuals. The variability is due to either a gain or lost of repeats during replication.

16. 16 9/21/2010 Microsatellite DNA These changes do not have any biological effect because the sequences do not code for any protein. An individual inherit one microsatellite from each parent. The chance of finding two individuals within the same population with the same DNA fingerprint is one in 105 - 108. In other words an individuals DNA fingerprint is almost as unique as his or her fingerprint. DNA Fingerprinting

17. 17 9/21/2010 Random Amplified Polymorphic DNA (RAPD) Another method widely used in characterization of DNA is RAPD. RAPD is often used to show relatedness among DNA populations. In this procedure arbitrary (random) primers are used during PCR to produce a fingerprint of the DNA. A single primer is used which must anneal in 2 places on the DNA template and region between the primers will be amplified. RAPD The primers are likely to anneal in many places on the template DNA and will produce a variety of sizes of amplified products. Amplified products are separated by agarose gel electrophoresis and visualized. If the samples have similar genetic make up then the pattern of bands on the gel will be similar and vice versa. This procedure is widely used to differentiate between different cultivars/var of the same plant. Issues to consider when using this procedure include reproducibility, quality of DNA, and several primers may have to be used.

18. 18 9/21/2010 RAPD Bacterial Biosensors Bacterial sensors can be used to test for environmental pollutants. Bacteria with bioluminescent are good candidates for pollutant sensors. In the presence of pollutants the bioluminescent decreases. The structural genes (luxCDABD) encodes the enzyme for bioluminescent was cloned into the soil bacteria Pseudomonas fluorescens. The cells that luminescence to the greatest extent and grew as well as the wild type were tested as pollutant sensors.

19. 19 9/21/2010 Bacterial Biosensors To screen water samples for pollutants (metal or organic) a suspension of P. fluorescens was mixed with the solution to be tested. After a 15 min incubation the luminescence of the suspension was measured. When the solution contained low to moderate levels of pollutants the bioluminescence was inhibited. The procedure is rapid, simple, cheap and a good screen for pollutants. Bacterial Bisensor