Fundamentals of Biotechnology

1. Fundamentals of Biotechnology Molecular Diagnostics

2. Molecular Diagnostics • The success of modern medicine and agriculture depends on the detection of specific molecules e.g. • Viruses • Bacteria • Fungi • Parasites • Proteins and Small Molecules • In water, plants, soil and humans.

3. 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.

4. Comparison of Methods Used to Diagnose Parasite Infections

5. Immunological Diagnostic Procedures • Immunological diagnostic procedures are often used to: • Test drugs • Monitor cancers • Detect pathogens Limitation if the target is protein part ( biochemical) • 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 testmolecule or organism to a solid support e.g. micro titer plate.

6. 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 antibodywhich will bind to the primary antibody. • The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. • Washto remove unbound antibody. • Addition of a colourless substratewhich will react with the secondary antibody to give a colour reactionwhich indicates a positive result.

7. ELISA

8. Antigens and Antibodies • Most antigens have many antigenic determinants or epitopes • Injecting antigens into mammal produces serum of polyclonal antibodies • Bind to many different epitopes and with different affinities • A uniform, high affinity, highly specific antibody preparation would be preferable • Monoclonal antibodies

9. Drawbacks of Polyclonal Abs • In diagnostics: • The amount of different Abs within a polyclonal preparation may be very from one batch to the next • they cant be used to distinguish between two similar targets • e.g. when the difference b.w the pathogenic (target) and Non Pathogenic one (non target) is single determined.

10. Monoclonal Antibodies • B Cell can’t be culture but their hybrid can. • Each B cell produces only one antibody (all of the molecules produced are identical) • A clone of B cells therefore all produce identical antibodies • Identify the proper clone and the culture of these cells produces a monoclonal antibody against the desired epitope

11. HAT Selection Cells obtain purines and pyrimidines by either of two ways • De novo biosynthesis requiring dihydrofolate reductase (DHFR) activity • Salvage requiring HGPRTase (hypoxanthine guanine phosphoribosyl transferase) for purines • Drug aminopterin inhibits DHFR forcing cells to utilize salvage pathways • HGPRTase (-) cells cannot salvage purines (hypoxanthine or guanine)

12. HAT Selection Procedure • HAT • Hypoxanthine • Aminopterin • Thymidine • Protocol • Immunize mouse with Ag • Isolate spleen (B cells) • Fuse with HGPRT- myeloma tumor cells • Select for actively dividing HGPRT+ cells on HAT medium

13. Screening for Monoclonal Antibody Production • HGPRT+ clones screened by immunoassay (use in ELISA protocol) • Clones that produce antibody binding to target antigen cultured and further characterized

14. Targets for Monoclonal Antibody-based Diagnostic Tests

15. DNA Diagnostic Systems • Presence of an organisms genetic material is a strong indicator of the presence of the organism or infectious agent • DNA Diagnostic Systems include: • DNA Hybridization • PCR • Restriction endonuclease analysis • RAPD (random amplified polymorphic DNA) • DNA fingerprinting

16. 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. • Nucleic Acid Hybridization diagnostic test has 3 critical elements: • Probe DNA, Target DNA, Signal detection.

17. DNA Hybridization • For DNA hybridization: • A probe is needed which will anneal to the target nucleic acid. • Attach the targetto a solid matrix e.g. membrane. • Denaturation ofboth the probe and target. • Add the denatured probe in a solution to the target. • If there is sequence homologybetween 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.

18. DNA Diagnostic Probes • DNA or RNA: long (>100 nts) or short (<50 nts): and chemically synthesis, Cloned intact gene, or isolated region of a gene • Useful for detection of parasites • Distinguish readily between subtypes • Distinguish readily between present and past infections (not necessarily easily done by some ELISA protocols) • PCR makes highly sensitive

19. Nonradioactive Detection Procedures • Chemiluminescent detection of bound DNA probe • Biotin-labeled nucleotides • Streptavidin (SA) binds biotin • Alkaline phosphatase conjugated to biotin also binds SA • AP cleaves small molecule releasing light

20. Detection by Fluorescent Dyes • Fluorescent dye attached to PCR primer(s) • PCR product now fluorescent-labeled • Detect following laser activation

21. Molecular Beacons • Probe • Palindromic region • Fluorophore • Quencher • Binding of probe to target sequence separates quencher from fluorophore • Laser activation for detection

22. Molecular Beacons • Molecular beacons with different fluorophores allow for simultaneous testing for multiple sequences

23. Detection of Heterozygotes • Use of multiple fluorophores and lasers

24. Detection of Malaria • Malaria is caused by the parasite Plasmodium falciparum. • The parasite infects and destroys red bloodcells. • Symptoms include fever, rashes and damage to brain, kidney and other organs. • Current treatment involves microscopic observationsof blood smears, which is labour intensive. • Other methods e.gELISAdoes not differentiate between past and present infection.

25. Detection of Malaria • A DNA diagnostic system would only measure current infection. • The procedure involves: • A genomic libraryof the parasite was screened with probes for parasitic DNA. • The probes which hybridized stronglywere tested further. • The probes were tested for their ability to hybridize to other Plasmodium specieswhich do not cause malaria and to human DNA.

26. 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) (188bp DNA present in multiple copies)

27. 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.

28. 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 cDNAcopy of the RNA of the virus. • This cDNA is used as a template to make dsDNA.

29. RT-PCR Diagnosis of HIV

30. Using PCR to Detect for HIV • Specific primers are used to amplify a 156 bp portion of the HIVgag 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.

31. 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 e.g. • In legal proceedings to identify suspects and clear others. • Paternity testing

32. 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 blottingto a membrane. • Membrane is hybridizedwith 4-5 different probes. • Detectionof hybridization.

34. Minisatellite DNA • After hybridization the membranes are stripped and reprobed. • The probes used are human minisatelliteDNA. • These sequences occur in the human genome as repeated sequences. • e.gATTAG….ATTAG….ATTAG…. • The length of the repeat is 9-40 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.

35. Minisatellite 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 uniqueas his or her fingerprint.

36. DNA Fingerprinting

37. Random Amplified Polymorphic DNA (RAPD) • Another method widely used in characterization of DNA isRAPD. • RAPD is often used to show relatednessamong 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.

38. RAPD • The primers are likely to anneal in many placeson the template DNA and will produce a variety of sizesof 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/varieties of the same plant. • Issues to consider when using this procedure include reproducibility, quality of DNA, and several primers may have to be used.

39. RAPD Analyses

40. RAPD

41. 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) (vibriofischeri) 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.

42. Bacterial Biosensors • To screen water samples for pollutants (metal or organic) a suspension of P. fluorescenswas mixed with the solution to be tested. • After a 15 min incubation the luminescence of the suspension was measured in luminometer. • 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.

43. Bacterial Bisensor

44. Restriction Digest Analysis(Molecular Diagnosis of Genetic disease) • Diagnosis of sickle cell anemia. • Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6thaa 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 oxygento supply these systems.

45. 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. • CvnIsite 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.

46. Detection of Sickle cell anemia by PCR

47. Cystic Fibrosis: • Autosomal recessive Common in Europe, • 500 mutation CFTR gene: (Cystic Fibrosis transmembrane conductance regulator) • Four the most common 81%.

48. 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).

49. 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.

50. PCR/OLA