1. DNA Technology in Human Identification Dr. Philip Beh Dr. G. Srivastava

2. Outline Identification Classification Individualisation Methods of Identification Development of Forensic DNA Forensic issues Interpretation of results Future

3. Identification Classify – Eg. Human or not? Human – Sex? Race? Age? Identification – Nationality? Identification – Locality? Identification – Identity (Individualisation!)

4. Human Identification How do we identify an individual? The human brain associates a name with an individual using a variety of information including physical appearance, mannerisms, voice and speech, etc.

5. Visual Identification Despite the complexity and accuracy of visual identification of an individual, it is well known that mistakes are common and in some circumstances unreliable and difficult.

6. Unreliable visual ID Time factors – aging, etc. Psychological and emotional factors – poor recall Lighting – inadequate or problematic lighting Physical changes especially in forensic work.

7. Individualisation One and only No other copy except for a genetic twin.

8. Other physical attributes Ear-shapes Lip prints Retinal scans Voice patterns (?) Handwriting (under scrutiny!)

9. Other means of individualisation Blood Blood groups HLA typing DNA DNA Fingerprinting DNA Typing

10. Development of DNA Fingerprinting 1985, Sir Alec Jeffries, described variable number tandem repeats (VNTR`s) and developed the technique restriction fragment length polymorphisms (RFLP) Briefly, it used restriction enzymes to cut the regions of the DNA surrounding the VNTR’s.

11. History First use in casework in the U.K. in 1985. First commercial labs in the U.S. in 1986 Used by the FBI in the U.S. in 1988. Used in Hong Kong in early 90’s. DNA-PCR technology used in Hong Kong in 1997.

12. Uniqueness Except for identical twins the DNA of an individual is unique. The number of different chromosomes that a child receives from parents are 2 46

13. Requirements for forensic casework Reliability of technique Reproducible results No laboratory error Security of test samples and results

14. DNA Polymorphisms Sequence polymorphism ….AGACTAGACATT….. ….AGATTAGGCATT….. Length polymorphism ….AATGAATGAATG…. ….AATGAATG…..

15. RFLP Restriction fragment length polymorphism A restriction enzyme is used to cut the DNA into fragments at specific points These fragments of different lengths are separated by an electric current. The fragments of interest are then radiolabelled by hybridisation.

16. RFLP Many RFLP systems are based on change in a single nucleotide. They are said to be diallelic Thus only two common alternative forms and three phenotypes, two homozygous and one heterozygous.

17. PCR Based Systems Length Polymorphisms STR kits - Short Tandem Repeats Sequence Polymorphisms PolyMarkers eg. DQ-alpha/A1 (HLA - DNA) Mitochondrial DNA Automated systems now available.

18. STR Short-tandem repeats Higher incidence of homozygotes, since the system is less polymorphic Several loci can be amplified Increase discriminating power with use of multiple probes.

19. Length Polymorphisms PCR used to amplify this. Much simpler than RFLP analysis because the DNA of interest already amplified. Bands stained directly Trend towards fluorescent detection and automated analysis.

25. Forensic use of DNA technologies Identification of small quantities of biological samples found, e.g. blood stains, semen, saliva stains, etc. Differentiation between origins of samples found. Linking and/or grouping of unknown sample.

26. Sources of DNA

27. Obstacles in forensic casework Small quantity of samples to work with. Contamination of samples. Poor preservation of material from which DNA is to be extracted. Eg. Contaminated stains, decomposition of tissues.

28. Complicating factors and forensic challenges Multiple contributors (sources) – mixed sample. Differential extraction Degradation Contamination Inhibition of enzymes Non-human DNA

29. DNA Extraction methods employed

33. Reverse Dot Process

36. Commonly used markers

37. CODIS Combined DNA Index System 1990 as a pilot project at the FBI Laboratory. Now in more than 100 public laboratories

38. Quality and standards DNA Advisory Board Quality assurance standards Laboratory Validation American Society of Crime Lab Directors Laboratory Accreditation Board European DNA Profiling Group Interpol European Working Party on DNA Profiling

39. Controls Monitoring for False Negatives False negatives arises from inhibitors. Safeguard against false negatives Positive controls – similar physiochemical properties and contains known DNA.

40. Controls Monitoring for False Positives False positives generally arises from contamination. Safeguards against false positives A negative control – undergoes the whole procedure, similar physiochemical properties except for genetic property. A blind control – undergoes all extraction, purification and amplification procedures, except that it does not contain any sample material. A no-template control – serves only for the amplification reagents and conditions. It contains every amplification reagents except DNA

41. Significance of results Three possible conclusions:- 1. Exclusion – they are different. 2. Inconclusive 3. Similar

42. Similarity Three possible scenarios:- 1. Sample from a common source 2. Coincidence 3. Accident (Error)

43. Frequency Estimate Calculations Hardy-Weinberg equilibrium There is a predictable relationship between allele frequencies and genotype frequencies at a single locus. This is a mathematical relationship that allows for the estimation of genotype frequencies even if the genotype has not been seen in an actual population survey.

44. Frequency Estimate Calculations Linkage equilibrium Defined as the steady-state condition of a population where the frequency of any multi-locus genotypic frequency is the product of each separate locus. This allows for the estimation of a DNA profile over several loci, even if the profile has not been seen in an actual population survey.

45. DNA evidence in Court This posed a problem due to a hosts of poorly researched and performed work and estimate calculations. It is now quite widely accepted and increasingly the frequencies are individualising e.g. 1 in several billions!!

46. Population Data Population data is required to obtain the various frequencies of occurences. Systems used require careful validation prior to use and also require internal and external controls for each test.

47. Future - Now Automation DNA databases Variant Repeats – approaches individualisation with just one or two loci More loci and marker systems SNP’s Quality control will continue to be an area of contention. More applications and more probes Faster automation, etc

49. DNA Databases Privacy issues Quality control of data Convicted samples vs. forensic case work samples

50. Ancient DNA Made possible by the availability of PCR The term ancient DNA now covers any bulk or trace DNA from a dead organism or parts of it, as well as extracorporeally encountered DNA of a living organism. Therefore any DNA that has undergone autolytic or diagenetic processes or fixation is considered “aDNA”

51. Reference Ancient DNA – Bernd Hermann & Susanne Hummel, Springer (1994) ISBN 0-387-94308-0

52. Mitochondrial DNA Advantage of having multiple copies present; therefore higher chance of detection in degraded material compared with nuclear DNA. Increasing use, different methodologies. About 4800 human mtDNA sequences available in forensic databases. An observed sequence is reported as the number of times an observed sequence is present in known databases and as such a frequency percentage is given. Possible to exclude 99%.

53. Mitochondrial DNA Discriminatory powers are still poor. Similarities may be seen in several individuals with similar maternal root. Useful in “population genetics” and in tracing the maternal line.

54. Y-Chromosome Increasing interest since it was sequenced. Y- chromosomes had about 6 of its 50 million DNA letters in palindromes. Have also been used to trace the “male line” in population studies Useful for working on mixed samples. May be key to “racial or regional characterisation”. Large numbers of commercial kits now available.

55. Future Low-copy numbers – risk of contamination high Racial – regional origins. Physical attributes from DNA Psychological/behavioral attributes Genetic predisposition for drug tolerance, for rare disorders, etc.

56. Recommended reading DNA Technology in Forensic Science – National Research Council (1992) ISBN 0-309-04587-8 The Evaluation of Forensic DNA Evidence – National Research Council (1996) ISBN 0-309-05395-1 An Introduction to Forensic DNA Analysis – K. Inman & N. Rudin. CRC Press (1997) ISBN 0-8493-8117-7 Forensic DNA Typing – Biology and Technology Behind STR Markers - John M. Butler. Academic Press (2001) ISBN 0-12-147951-X

57. Recommended reading Rutty GN et al “Contamination of mortuary instruments and work surfaces by human DNA; A significant problem in forensic practice?” Int. J Legal Med 2000; 114: 56-60 B.S. Weir “Population genetics in the forensic DNA debate.” Proc. Natl. Acad. Sci. USA Vol 89 pp 11654-11659, December 1992 John Whitfield “Y chromosome sequence completed” Nature Science Update 19 June 2003.