DIAGNOSTICS AND BIOMARKERS

Transcription

1 DIAGNOSTICS AND BIOMARKERS

2 MODERN TECHNOLOGIES FOR DIAGNOSTICS TYPES ANALYTICAL PARAMETERS USE PARAMETERS

3 TYPES BASED ON

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6 Molecular DiagnosUcs The success of modern medicine depends on the detecuon of specific molecules e.g. Viruses Bacteria Fungi Parasites Proteins In water, plants, soil and humans. 6

7 CharacterisUcs of a DetecUon System A good detecuon system should have 3 qualiues: SensiUvity Specificity Simplicity Sensi&vity 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 posi&ve result for the target molecule only Simplicity: the test must be able to run efficiently and inexpensively on a rouune basis 7

8 Immunological DiagnosUc Procedures Immunological diagnosuc procedures are o^en used to: Test drugs Monitor cancers Detect pathogens ELISA (Enzyme Linked Immunosorbent Assay) This involves the reacuon of an an&body with an an&gen and a detec&on system to determine if a reacuon has occurred. ELISA involves: Binding of the test molecule or organism to a solid support e.g. micro Uter plate. 8

9 ELISA AddiUon of a specific an&body (primary anubody) which will bind to the test molecule if it is present. Washing to remove unbound molecules. AddiUon of secondary an&body which will bind to the primary anubody. The secondary anubody usually has a7ached to it an enzyme e.g. alkaline phosphatase. Wash to remove unbound anubody. AddiUon of a colourless substrate which will react with the secondary anubody to give a colour reac&on which indicates a posiuve result. 9

10 ELISA 10

11 DetecUon of HIV 11

12 DNA DiagnosUc Systems DNA HybridizaUon PCR RestricUon endonuclease analysis RAPD (randomly amplified polymorphic DNA) DNA fingerprinung 12

13 DNA HYBRIDIZATION Oldest method Not as useful for closely related DNAs PCR is more useful Heating the DNA solution above a characteristic temperature can separate the two strands of a double helix. That temperature is called the melting temperature, abbreviated T m.

14 DNA HybridizaUon A probe is needed which will anneal to the target nucleic acid ACach the target to a solid matrix, e.g., membrane Denatura&on of both the probe and target Add the denatured probe in a soluuon to the target If there is sequence homology between the target and the probe, the probe will hybridize or anneal to the target Detec&on of the hybridized probe, e.g., by autoradiography, chemiluminsence or colorimetric 14

15 DetecUon of Malaria Malaria is caused by the parasite Plasmodium falciparum. What is a parasite? infects and destroys red blood cells Symptoms include fever, rashes and damage to brain, kidney and other organs Current treatment involves microscopic observa&ons of blood smears, which is labor intensive. Other methods, e.g, ELISA does not differenuate between past and present infecuon Why? 15

16 DNA DIAGNOSTIC FOR MALARIA Only measure current infec&on. (Why?) PROCEDURE: Screen genomic library (hybridizauon) with probes for malaria DNA Strongly hybridizing probes tested further: Which are best? Eliminate those that hybridize to other Plasmodium species which do not cause malaria or to human DNA 16

17 DetecUon of Malaria Probes which hybridized to P. falciparum = diagnosuc tool 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 (gastroenterius) Trypanosoma cruzi (Chagas disease) 17

18 Polymerase Chain ReacUon PCR uses 2 sequence- specific oligionucleo&de primers to amplify the target DNA The presence of appropriate amplified size fragment confirms presence of target. Specific primers are now available for the detecuon of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi. 18

19 RT- PCR for VIRAL RNA, e.g., HIV RT- PCR (reverse transcriptase PCR) HIV has ssrna genome. Lyse plasma cells from the potenually infected person to release HIV RNA genome. Precipitate RNA using isopropanal Make cdna (ssdna) copy of RNA using reverse transciptase PCR amplify cdna with Taq DNA polymerase using 2 primers (short regions of DNA) from a region of the genome, dntps, ions 19

20 DNA METHODS widespread and innumerable uses diagnose geneuc diseases DNA fingerprinung find microorganisms, study human evoluuon and migrauon clone DNA of EgypUan mummy establish paternity or biological relauonships Etc.

21 DNA HybridizaUon Bacterial and viral pathogens may be pathogenic because of the presence of specific genes or sets of genes GeneUc diseases o^en are due to muta&ons or absence of parucular gene or genes These genes (DNA) can be used as diagnosuc tools This involves using a DNA probe during DNA hybridiza&on What is a DNA probe? How does DNA hybridizauon work? 21

22 RT- PCR Diagnosis of HIV 22

23 Using PCR to Detect for HIV Specific primers are used to amplify a 156 bp poruon 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 prognosuc tool to determine viral load. This method can also be used to determine the effecuveness anuviral therapy. (Brock Biology of Microorganisms 9 th ed. pg ). 23

24 DNA FingerprinUng (RFLP) RFLP = Restric&on Fragment Length Polymorphism Regular fingerprinung analyses phenotypic traits. DNA fingerprinung analyses genotypic traits. DNA fingerprinung (DNA typing) is used to characterize biological samples e.g. In legal proceedings to idenufy suspects and clear others. Paternity tesung 24

25 DNA FingerprinUng (RFLP) The procedure involves: CollecUon of sample e.g. hair, blood, semen, and skin. Examina&on of sample to determine if there is enough DNA for the test. The DNA is digested with restric&on enzymes. Digested DNA is separated by agarose gel electrophoresis. DNA is transferred by Southern bloong to a membrane. Membrane is hybridized with 4-5 different probes. Detec&on of hybridizauon. 25

26 Microsatellite DNA A^er hybridizauon 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 9-40 bases occurring Umes. The microsatellites have different length and numbers in different individuals. The variability is due to either a gain or lost of repeats during replicauon. 26

27 Microsatellite DNA These changes have no biological effect because do not code for protein Inherit one microsatellite from each parent The chance of finding two individuals within the same populauon with the same DNA fingerprint is one in In other words an individual s DNA fingerprint is almost as unique as his or her fingerprint 27

28 Random Amplified Polymorphic DNA (RAPD) RAPD is o^en used to show relatedness among DNA populauons 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 28

29 RAPD used to determine similarity Primer(s) anneal in many places variety of sizes of amplified DNA products Products separated by agarose gel electrophoresis and visualized If similar geneuc make up then band pa7erns are similar Uses: differenuate between different plant culuvars/varieues Compare samples in a disease outbreak Issues: reproducibility, DNA quality, and number of primers needed (o^en > 1) 29

30 RAPD Which primer is useful? 30

31 The example is for plants. What might be an example for people?

32 RestricUon Digest Analysis EXAMPLE: Diagnosis of sickle cell anemia Sickle cell anemia is a geneuc disease caused by a single nucleoude change in the 6 th aa of the β chain of hemoglobin A (normal) glutamic acid and S (sickle) valine In the homozygous state SS red blood cells are irregularly shaped Result = progressive anemia and damage to heart, lung, brain, joints and other organ systems Mutant can t carry enough oxygen to supply these systems 32

33 Diagnosis of Sickle Cell Anemia The single mutauon in hemoglobin changes the restricuon pa7ern of the β globin gene abolishing a CvnI site. CvnI site CC TNAGG (N = any nt) Normal DNA sequence CCTGAGG (A) Mutant DNA sequence CCTGTGG (S) Use two primers which flank mutant region of β globin gene during PCR to amplify this Digest PCR products CvnI and separate them using agarose gel electrophoresis 33

34 sc DetecUon of Sickle cell anemia by PCR 34

35 PCR/OLA Like sickle cell anemia many geneuc diseases are caused by mutant genes Many caused by a single nucleoude (nt) change in the wild type gene A single nt change can be detected by PCR/OLA ( oligonucleoude ligauon assay) E.g. The normal gene has A at nt posiuon 106 and mutant has a G 35

36 PCR/OLA Synthesize 2 short oligonucleoudes (oligos) Oligo 1 (probe x) complementary to wild type having A at 106 (3 end) Oligo 2 (probe y) has G at 107 (5 end) Label them differently Incubate both probes with PCR amplified target DNA Add DNA ligate: two probes will only ligate if the two probes are perfectly aligned (as in the wild type). 36

37 PCR/OLA 37

38 PCR/OLA To determine if the mutant or wild type gene is present it is necessary to detect for ligauon. Probe x is labeled at 5 end with bioun Probe x is labeled at 5 end with digoxygenin. 38

39 PCR/OLA Digoxygenin serves as an anubody binding indicator. A^er washing a colourless substrate is added. If a coloured substrate appears this is indicauve that the bioun probe (x) ligated to the dioxygenin probe (Y) and that the wild type gene is present. 39

40 PCR/OLA 40

41 PCR/OLA Synthesize 2 short oligonucleoudes (oligos) Oligo 1 (probe x) complementary to wild type having A at 106 (3 end). Oligo 2 (probe y) has G at 107 (5 end). Incubate both probes with 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. 41

42 END