DNA Technology in Human Identification

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 246

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”