MOLECULAR ASSAYS FOR GENETIC TESTING

1. MOLECULAR ASSAYS FOR GENETIC TESTING

2. Human Genetic Testing • Human genetic testing serves four main purposes: a. Prenatal diagnosis b. Newborn screening • Carrier (heterozygote) detection • Disease predisposition

3. KARYOTYPING Karyotyping of fetal cells from amniotic fluid or chorionic villi –gross chromosomal defects, trisomies etc

4. Trisomy 21 karyotype

5. I - Molecular Hybridization For the detection of a specific DNA sequence in a heterogeneous mixture of DNA (e.g. whole genomic DNA etc)

6. Probes Labeled, single stranded DNA complementary to the target DNA The label may be radioactive (P32) or non radioactive (fluorescent chromophores or biotin)

7. General scheme for hybridization assays Labeled probe Heterogeneous mix of DNA Probe binds target DNA

8. Probes Target DNA sequence known Short oligonucleotide probes,15-30 bases, complementary to part of the target sequence Protein sequence known Several oligonucleotide probes constructed using the genetic code, to locate the target sequence

9. Hybridization- the flow chart Denature target DNA Immobilize on insoluble matrix Expose to labeled probes Allow time for hybridization Wash away unbound probe Detect for the presence of label

10. APPLICATION Mr. and Mrs. are expecting their second child. They know that sickle cell anemia runs in both of their families. They want to know whether this child could be affected. Neither they nor their 10-year-old daughter have shown any symptoms of the disease. They decide to have DNA tests to determine the status of the fetus, as well as to find out whether they in fact are carriers of the disease gene. They send their samples to a DNA diagnostic facility where their samples are analyzed using the allele specific oligonucleotide (ASO) probe technique.

11. Allele specific oligonucleotide probe - detection of bs-globin mutation SICKLE CELL ANEMIA AT mutation in the b-globin gene converts glutamate  valine at position 6 of the protein Normal CT CCT GAG GAG AAG TCT GC GA GGA CTC CTC TTC AGA CG Mutant CT CCT GTG GAG AAG TCT GC GA GGA CAC CTC TTC AGA CG

12. Allele specific oligonucleotide probe - detection of bs-globin mutation Two probes are designed CT CCT GTG GAG AAG TCT GC One complementary to the normal DNA sequence CT CCT GAG GAG AAG TCT GC Normal probe GA GGA CTC CTC TTC AGA CG The other complementary to the mutant DNA sequence Mutant probe GA GGA CAC CTC TTC AGA CG

13. Allele specific oligonucleotide probe - detection of bs-globin mutation Normal Probe Mutant Probe Homozygous normal AA Heterozygous carrier AS = PROBEHYBRIDIZES Homozygous mutant SS = PROBE DOES NOT HYBRIDIZE

14. Allele-specific PCR Normal CTCCTGAGGAGAAGTCTGCNNNNNNNNNN Mutant CTCCTGTGGAGAAGTCTGCNNNNNNNNNN Template DNA + dNTPs+MgCl2+Taq Polymerase, primers Template DNA + dNTPs+MgCl2+Taq Polymerase, primers If Normal (A) amplification takes place If Mutant (T) amplification does not take place If Normal (A) amplification does not take place If Mutant (T) amplification takes place

16. APPLICATION A 32-year-old female presents to your clinic with concerns over a recently detected right breast lump. A biopsy is performed and reveals an intraductal carcinoma. She is invited to participate in an experimental study that is being carried out to help direct future treatment protocols and define new drug targets for breast cancer. The researcher explains to her that DNA microarrays (DNA chips) will be used to study the differences in the gene expression profiles of tumor versus normal cells. After considering all the pros and cons she gives her informed consent and allows her tissue samples to be used.

17. Microarrays/DNA chips Microarrays/DNA chips small, solid supports onto which DNA sequences from thousands of different genes are immobilized • Expression analysis • Genotyping • SNP analysis • Mutation detection

18. Expression analysis using microarrays

19. Applications of DNA microarrays • Cell specific expression • Gene regulation • Tumor profiling • Genetic variation • Microbial strain identification • Drug testing

20. APPLICATION Mr. and Mrs. JD are expecting their first child. Mr. JD’s uncle had died of cystic fibrosis (CF) and they recently learnt that a distant cousin of Mrs. JD has also been diagnosed with CF. They are worried that they might be carriers for the disease. Their doctor suggests an amniocentesis to detect if their unborn child has CF or is a carrier. They feel that an amniocentesis is an invasive and risky procedure and decide that they first want to be tested themselves to see if they are carriers for the disease. If they learn that they both are carriers, they would like to go through with the amniocentesis to see if their child is affected. The most common mutation, accounting for about 75% of CF cases, is called delta F508 and can be screened using the AFLP (amplified fragment length polymorphism) technique.

21. Human genomic DNA contains restriction sites for restriction enzymes • Very often a mutation can result in the creation of destruction of a restriction site e .g. GAATTC = restriction site for EcoRI Destruction of restriction site GAATTC GATTTC or Creation of restriction site GGATTC GAATTC

22. RFLPS typed using PCR MspI Forward primer Reverse primer PCR Amplification Restriction digestion with MspI (Δ508F lead to loss of MspI site) Run on agarose gel

23. 400 bp Forward primer Reverse primer 300 bp 100 bp

24. APPLICATION Mr. and Mrs. SZ just had their first child. The phenylketonuria (PKU) blood test performed at birth indicated a high level of phenylalanine in the blood. The physician suggests a follow-up DNA test immediately to confirm the PKU diagnosis. None of the mutations known to cause PKU in the phenyalanine hydroxylase gene is picked up by the standard testing methods. The lab therefore decides to carry out DNA sequencing of the child’s sample to check for the presence of a novel gene mutation

25. DNA sequencing Characterization of DNA sequence is through dideoxy DNA sequencing (Sanger method)

26. Automated DNA sequencing

27. A DNA sequence

28. APPLICATION The body of an unidentified young woman is found stuffed in a sack in a forest. She has multiple stab wounds and her face has been mutilated beyond recognition. The parents of a girl, who had reported their daughter missing a few days ago, are asked to provide blood samples for DNA Profiling to establish if the body may be of their daughter. The sack is which the girl was found is found to have several hair on it which do not belong to the girl. They are collected as forensic evidence.

29. DNA Fingerprinting by RFLPs (1987-mid 1990s) • DNA Fingerprinting by RFLPs (Restriction Fragment Length Polymorphisms) was developed in the early 1980s by Sir Alec Jeffreys • It made use of genetic variation in the distance between restriction enzyme sites - Due to the presence of VNTRs (Variable Number of Tandem Repeats) • Power of discrimination was in the range of 106-108 for a six probe analysis

30. Variable Number of tandem repeats/ Short tandem repeats (STRs) Individual 1 7 repeats Individual 2 8 repeats Individual 3 9 repeats

31. DNA Fingerprinting Person 1 Person 2             restriction fragments 1 2

32. The Steps: DNA Fingerprinting DNA prepared (from crime scene samples and suspects) Cut with restriction enzymes Restriction fragments separated using gel electrophoresis Transferred to a nylon membrane Exposed to radiolabelled probe that binds to its complimentary DNA fragments Photographic image obtained

33. The Colin Pitchfork Case FIRST EXONERATION AND CONVICTION BASED ON DNA EVIDENCE • Two young women were raped and murdered in Narborough, England (1983 and then in 1987) • Police contacted Alec Jefferys for DNA fingerprinting • The first suspect (who had confessed) was excluded • 5,000 local men were then asked to provide blood/ saliva samples • CP convicted in 1988

34. Human Identity Testing • Crime scene investigation -- matching suspect with evidence • Paternity testing -- identifying father • Missing persons investigations --whose body • Mass disasters -- putting pieces back together • Inheritance Claims – who gets the money • Historical investigations • Military DNA “dog tag”

35. DNA profiling requires a reference sample • A DNA profile on its own has NO context • DNA profiling works by comparison • Crime scene evidence compared to suspect (forensics) • Child compared to alleged father (paternity) • Victim’s remains compared to a biological relative (mass disaster ID) • Soldier’s remains compared to a reference sample (Armed Forces ID)

36. Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue

37. DNA fingerprinting by RFLPs: the downside • RFLP testing requires a relatively large amount of HMW DNA (50-250ng = thousands of cells) • Not ideal for forensic evidence, in which small, degraded samples are common

38. PCR to the Rescue!! • Polymerase Chain Reaction = Molecular Xeroxing • Series of cycles of three successive steps, carried out in a Thermal Cycler, “amplify” the desired DNA fragment(s) 5 cycles of PCR = 64 copies of DNA 40 cycles of PCR = 1.099 x 1012 copies of DNA!!

39. The Steps: STR Typing Extract and purify DNA (from crime scene samples and suspects) Carry out PCR Run PCR product on a genetic analyzer Assign genotypes

40. Singleplex PCR Forward primer DNA + Reverse primer + dNTPs+MgCl2+Taq Polymerase

41. Multiplex PCR Locus 1 Locus 2 DNA + Primers Locus 3 Locus 4 + dNTPs+MgCl2+Taq Polymerase Simultaneous amplification of four locations on a DNA template

42. DNA sequencer ABI 310 Genetic Analyzerseparates amplified DNA

43. Detector Window ABI 310 Genetic Analyzer: Capillary Electrophoresis • Amplified STR DNA injected • Electric current applied • DNA pulled towards the positive electrode • DNA separated out by size: • Large STRs travel slower • Small STRs travel faster • Color of STR detected and recorded as it passes the detector

44. PATERNITY TESTING FATHER MOTHER GIRL

45. Advantages of STR Typing • < 1ng of DNA is required to type 13-15 STR loci • Can be processed within 24 hrs • Relatively degraded DNA samples can be used • Power of discrimination ranges from 1014-1023. World population is 6 x 109

46. APPLICATION • A 60 year old heroine addict presents at the OPD with a history of repeated episodes of flu, fever, malaise, and maculopapular rash. He reveals that has been sharing needles indiscrinately with other addicts. Laboratory investigations include ELISA for HIV.

47. Enzyme-linked Immunobsorbent Assay (ELISA) • The ELISA method is a diagnostic test used to detect antibodies or proteins associated with a specific clinical conditions. • The basic principle of an ELISA is to use an enzyme to detect the binding of antigen (Ag) antibody (Ab). • The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. • An ELISA can be used to detect either the presence of Ags or Abs in a sample, depending on how the test is designed. • The development of color in an ELISA test indicates a positive result.

48. Coat plate with capture mAb (in this case specific for HIV antigens) Add test samples andstandard Add biotinylated (labelled) detection mAb Add streptavidin-enzyme Add chromogenic substrate for colordevelopment

49. Indirect ELISA Enzyme-linked immunobsorbent assay (ELISA) Colorless substrate Colored product