Molecular diagnostic techniques

Molecular diagnostic techniques - Dr Meghana P

1. MOLECULAR DIAGNOSTIC TECHNIQUES Dr. Meghana P PG in Pathology

2. Common methods used in molecular biology, biochemistry, genetics and biophysics which generally involve manipulation and analysis of DNA, RNA and Proteins . These techniques have utility in virtually every area of diagnostic pathology – neoplastic disorders, infectious diseases, inherited conditions and identity determination. MOLECULAR DIAGNOSTIC TECHNIQUES

3. TYPES Amplification techniques – PCR and its modifications Blotting techniques – Southern blot , Dot blot, RFLP Northern blot Western blot Hybridization techniques – In situ hybridization FISH and its modifications Microarray

4. SAMPLE Virtually all pathologic material is suitable. Formalin fixed. Paraffin embedded. Small volumes of whole blood or glass slides.

5. PRINCIPLE

6. PRE REQUISITES Restriction endonucleases Vectors Probes

7. RESTRICTION ENDONUCLEASES Molecular scissors. Cleave the DNA at specific sequence. Produce sticky or blunt ends.

8. VECTORS Carrier of DNA molecule to which the fragment of DNA of interest is attached.

9. TYPES OF VECTORS VECTOR kbp Plasmids 0.01 - 10 Phages 10 - 20 Cosmids 30 - 50 BAC 50 - 250 YAC 500 - 1000

10. PROBES Pieces of DNA or RNA labelled by various techniques to detect a complementary sequence. Uses: Detect DNA and RNA Search libraries

11. LABELLING OF DNA PROBES Radioactive labels – 32 P Nonradioactive labels – Biotin label Fluorescent labels

12. AMPLIFICATION TECHNIQUES

13. POLYMERASE CHAIN REACTION - PCR Developed by Kary Mullis in 1984. Test tube method. Amplifies a selected DNA fragment.

14. PRE REQUISITES OF PCR DNA Primers Taq polymerase dNTPs

15. PRIMERS 2 set of primers Generally 20-30 nucleotides long. Synthetically produced. 40-60% GC content preferred.

16. PCR CYCLE STEP 1: Denaturation of DNA 94 C

17. STEP 2: Annealing with primers 55 C PCR CYCLE

18. STEP 3: Extension of the primer 72 C, enzyme and substrate PCR CYCLE

19. END OF 1 st PCR CYCLE

20. MODIFICATIONS OF PCR RT – PCR Real time PCR Nested PCR Multiplex PCR Inverse PCR Asymmetrical PCR Anchored PCR Immuno PCR Competitive PCR In situ PCR Single cell PCR Ligase chain reaction (LCR) Amplification refractory mutation system (ARMS) Q - beta replicase Nucleic acid sequence – based amplification (NASBA)

21. REVERSE TRANSCRIPTION PCR (RT-PCR) Amplifies DNA from RNA. Reverse transcriptase enzyme is used. Thermostable - Tth polymerase.

22. REAL TIME / QUANTITATIVE PCR REAL TIME PCR TRADITIONAL PCR DETECTION while the reaction is occurring. at the end point of the reaction. DATA COLLECTION after each cycle of the PCR. only at the end of 20-40 cycles. Fluorescent reporter signal

23. NESTED PCR 2 sets of primers - outer and inner. Increases both sensitivity and specificity of the assay.

24. MULTIPLEX PCR Uses more than one pair of primers. Amplification of many targets in one reaction.

25. ADVANTAGES Automated Rapid Reliable Contained Sensitive Defined

26. LIMITATIONS Sequence i nformation Error during amplification Sensitivity to inhibitors Contamination

27. BLOTTING TECHNIQUES

28. BLOTTING TECHNIQUES The analytical techniques used for the identification of specific DNA, RNA or a Protein from thousands of each.

29. TYPES OF BLOTTING TECHNIQUES Southern Blot – DNA Northern Blot – RNA Western Blot – Proteins

30. SOUTHERN BLOTTING Professor Sir Edwin Southern , developed this method in 1975. Principle: Based on specific base pairing rule of complementary nucleic acid strands.

31. SOUTHERN BLOTTING

32. DOT BLOT TECHNIQUE Blotting to nitrocellulose membrane is avoided. Sample is directly applied to slots on a specific blotting apparatus containing nylon membrane. Slot Blot.

33. RESTRICTION FRAGMENT LENGTH POLYMORPHISM Inherited difference in the pattern of restriction map produced by the digestion of a specific restriction endonuclease . DNA variations that result in RFLP - Single Nucleotide Polymorphism (SNP) Variable Number Tandem Repeats (VNTR) Mutations

34. SNP: - Occur at intervening sequences. - Clinically harmless. VNTR: - Short sequences of DNA that are repeated multiple times. Mini – satellite DNA sequences. Mutations: - Point mutation – sickle cell anemia

35. USES OF RFLP Tracing chromosomes from parent to offspring. Prenatal diagnosis of diseases – sickle cell anemia , phenylketonuria . Medicolegal uses. Detect point mutation, insertion and deletion.

36. RESTRICTION FRAGMENT LENGTH POLYMORPHISM

37. NORTHERN BLOTTING Developed by James Alwine and George Stark in 1979. Principle: RNA – DNA hybridization technique.

38. NORTHERN BLOTTING cDNA

39. WESTERN BLOTTING Principle : Antigen antibody interaction. Antigen – protein of interest Antibody – probe

40. WESTERN BLOTTING

41. HYBRIDISATION TECHNIQUES

42. H YBRIDIZATION target DNA probe denaturation hybridization Formation of a duplex between two complementary sequences.

43. IN SITU HYBRIDISATION Metaphase chromosomes Radioactive probe

44. FLUORESCENT IN SITU HYBRIDISATION (FISH) Metaphase chromosomes Fluorescent probe Very sensitive

45. TYPES OF FISH PROBES Centromeric (satellite) probe s Whole chromosome painting probes Locus specific probe s

46. Fluorescent Microscope Camera Filters FISH Analysis Software

47. APPLICATIONS OF FISH Detection of aneuploidy . Demonstration of subtle micro deletions. Detection of complex translocations. Analysis of gene amplification – HER2/ neu N- Myc Mapping newly isolated genes.

48. MULTICOLOR FISH Spectral karyotyping . Combination of five fluorochromes are used. By appropriate computer generated signals, the entire human genome can be visualized.

49. MICROARRAY TECHNIQUE DNA chip, next generation sequencing. Contain thousands of known immobilized DNA sequences. The fluorescently tagged sample to be sequenced is added. Genotyping – single stranded genomic DNA is added. Gene expression – cDNA is added.

50. MICROARRAY

51. ARRAY CGH Array-based Comparative Genomic Hybridisation. (Array - Based Comparative Genomic Hybridization)

52. APPLICATIONS OF MOLECULAR DIAGNOSTIC TECHNIQUES

53. HEMATOLOGICAL MALIGNANCIES DISEASE TRANSLOCATION CML, ALL t (9,22) Follicular non – Hodgkin’s lymphoma t (14,18) Anaplastic large cell lymphoma t (2, 5) Acute non lymphoblastic leukemia (FAB M2) t (8,21) Acute promyelocytic leukemia (FAB M3) t (15,17) Acute myelomonoblastic leukemia with abnormal eosinophils (FAB M4E) inv (16) Pre B – cell acute lymphoblastic leukemia t (1,19)

54. t(9,22) Inversion

55. NON HEMATOLOGICAL MALIGNANCIES DISEASE TRANSLOCATION Ewing’s sarcoma t (11,22) Peripheral neuroectodermal tumor t (21,22) Desmoplastic small round cell tumor t (11,22) Alveolar rhabdomyosarcoma t (2,13), t (1,3) Clear cell carcinoma t (12,22) Myxoid liposarcoma t (12,16)

56. EWSR1 rearrangement in Ewing’s sarcoma Normal Abnormal (translocation)

57. INFECTIOUS DISEASES Microorganisms detected by molecular techniques are – Viruses – HSV, HHV 6, EBV, CMV, HBV, HCV, HIV - 1 and 2, HTLV – 1 and 2 Bacteria – Mycobacterium, Neisseria , Helicobacter, Borrelia , Chlamydia Parasites – Toxoplasma , Trypanosoma , Plasmodium, Leishmania Fungi – Candida

58. IDENTIFICATION OF NEW ORGANISM Rochalimaea henselae – Bacillary angiomatosis Ehrlichia chaffeensis – Human ehrlichiosis Influenza A (subtype H1N1) – 2009 flu pandemic Zika virus – Zika fever endemic

59. INHERITED GENETIC DISEASES Albinism α -1 antitrypsin deficiency Ataxia telangiectasia Cystic fibrosis Familial hypercholesterolemia Fragile X syndrome Hemophilia A and B Osteogenesis imperfecta Mucopolysaccharidoses Sickle cell anemia Thalassemia vWD

60. IDENTITY DETERMINATION Transplantation – Tissue antigen matching Tracking engraftment Forensic pathology Parentage testing Twin zygosity Military usage of DNA dog tags Specimen mix-ups

61. OTHERS Production of proteins in abundance Gene therapy Industrial applications Agricultural applications Transgenesis Evolution

62. ETHICAL IMPLICATIONS Laboratory genetic manipulations may create certain new pathogens. Biological contamination. Detect disease susceptibility in clinically disease-free individuals. Human Genome Project is addressing these issues through its ELSI component.

63. SUMMARY The molecular techniques has revolutionized the application of molecular biology to medical and agricultural sciences that has immensely benefited mankind. An abundant production of DNA in a test tube has been successfully achieved by PCR. DNA probes are now available for the detection of certain genetic disorders. The human genome contains about 100,000 genes. Through the application of molecular techniques, it is expected that the entire sequence of human genome will be known soon.

64. REFERENCES Anderson: Diagnostic molecular pathology.199-222,2009. U.Satyanarayana , U. Chakrapani , Biochemistry, Arunabha Sen Books Pvt. Ltd., 2010. Robert K. Murray, Daryl K. Granner , Peter A. Mayer, Victor W. Rodwell ., Harper’s Illurstrated Biochemisry , Lange Medical publications, 2005. George J.N, Rana D.S, and Peter A.D. Diagnostic molecular pathology: current techniques and clinical applications. 2003 Oct; 16(4): 379–383. Min T, Swansbury J. Cytogenetic studies using FISH: background. Methods Mol Biol. 2003;220:173–191.

65. THANK YOU