Non-human DNA Testing

Transcription

1 Non-human DNA Testing

2 Finish up mtdna first

3 Different Laboratories Currently there are no commercial kits for mtdna genotyping Studies have proven mtdna profiles from different labs can be compared Standard Reference Materials (SRM) Positive Controls With know sequences Any negative controls show product Everything must be thrown out

4 Reporting sequence Do not report the entire sequence Instead only report the exact locations where the sequence differs from the CRS Cambridge Reference Sequence (rcrs) Report differences as: Base pair position Followed by base present (A, G, T, C) Example C

5 Interpreting mtdna Profile Three possible interpretations of mtdna profile for Forensics: 1. Exclusion Cannot have originated from same sample 2. Inconclusive Insufficient distinguishing power to identify a match or not 3. Failure to exclude Sample may be from same source, or any maternal relative of source

6 Interpreting mtdna Profile Failure to exclude: Sample may be from same source, or anyone with same maternal lineage Cannot determine Inclusion like you can with nuclear STRs Why not? mtdna profiles are useful for excluding a suspect, but not powerful enough to identify a unique match

7 Likelihood of mtdna Profile Mathematical approach used to calculate the likelihood of seeing a mtdna profile 1. Counting method p = X(# this exact profile)/n (# total profiles) Depends on the population the suspect belongs to Also on the population database used Larger the size of the database more accurate likelihood will be Like Y-STR; only qualitative

8 Interpretation of mtdna profiles Heteroplasmy More than one haplotype of mtdna within same individual Sample Mixtures Almost impossible to handle with mtdna Nuclear pseudogenes Genes in the nucleus as well as within mtdna Paternal leakage Incredibly rare situation where sperm donates some mitochondria to zygote

9 Heteroplasmy When different mtdna haplotypes exist within the same individual Sort of like being heterozygous Only caused by mutations rather than inheriting different alleles Certain hotspots exist within hypervariable regions Normally a single individual will carry one base pair heterozygous, not usually any more

10 Heteroplasmy Three types: More than one haplotype within one tissue Different haplotypes in different tissues Heteroplasmic in one tissue type; homoplasmic in another tissue type All will complicate mtdna analysis Must have more than 3 base pairs different before can exclude mtdna profile

11 Sample Mixtures Added complication of heteroplasmy And difficulties with sequencing when strands do not align Almost impossible to interpret mixed samples using mtdna If three or more bases are found to be heterozygous the sample is considered a mixture and analysis may be stopped

12 Nuclear Pseudogenes Certain mtdna genes exist in nucleus as well with different sequence Do not make functional protein Therefore consider pseudogenes If the pseudogene is amplified rather than the mtdna gene may complicate analysis Rarely cause a problem in real life due to initial copy number differences

13 Complications with mtdna: 1. Heteroplasmy More than one haplotype of mtdna within same individual 2. Sample Mixtures Almost impossible to handle with mtdna 3. Nuclear pseudogenes Genes in the nucleus as well as within mtdna 4. Paternal leakage Incredibly rare situation where sperm donates some mitochondria to zygote

14 Common mtdna profiles Depends on the population Some of the haplotypes are very common More than 10 % Therefore not very informative for forensic analysis like blood types Again mtdna and Y-STRs are more useful when they exclude a sample Often cannot provide unique profile alone

15 Resolving Most Common Types Trying to find new variations that separate the most common profiles apart Make each profile more informative Add the following additional markers to the hypervariable sequences: SNPs Sequencing larger region of mtdna Restriction fragments

16 Population Databases Important for estimating likelihood of seeing a given mtdna profile Gather as much information as possible High quality sequences exist for over 4000 unrelated individuals Different ethnic backgrounds Different countries All present within CODIS database

17 Now: Non-Human DNA Testing

18 Non-Human DNA Vast majority of forensic analysis is done on human DNA However it is not the only source of DNA present at a crime scene: Animal DNA Insect DNA Plant DNA Microbial DNA

19 Uses of Non-Human DNA Animal DNA: May be used to place a suspect at the scene Link suspect to victim Link victim s body to different crime scene Plant DNA: Rare plants may narrow down possible search areas Link suspect to scene Prove that victim s body was moved

20 Use of Non-Human DNA Insect DNA: Link suspect to crime scene Prove that victim s body was moved Calculate time of death Microbial DNA: Calculate time of death Aquatic microbes provide evidence of fresh or salt water involvement Bioweapons

21 Domestic Animals Over 64 million homes in US have pets Many of these pets shed hair: Especially cats and dogs These hairs may link a suspect to a victim Or link suspect to a crime scene Either because suspect picked up victim s pet s hair Or suspect left his pet s hair at scene

22 Animal DNA Three Types of animal DNA evidence: 1. Animal as witness 1. Animal DNA connecting suspect to victim 2. Connecting suspect to crime scene 2. Animal as victim 1. When an animal is illegally harmed 3. Animal as perpetrator 1. When an animal attacks a human

23 Hair Transfer Animal as witness Study has been done to stimulate amount of hair that may be transferred Hundreds of hairs may be transferred between suspect and victim and scene Authors of study felt it was almost impossible to enter a home with a pet without being contaminated by pet hair

24 Other Animal DNA Animal as victim If an animal is abused, killed or stolen DNA testing can identify remains Prove that animal was killed illegally Animal as perpetrator If animal attacks a human DNA testing can be done to be sure wrong animal is not needlessly destroyed

25 Cat DNA 18 Autosomes and XY sex chromosomes Panel of multiplexed STRs called MeowPlex 11 different STRs and gender Discrimination from MeowPlex is actually very high Depends on breed of cat Hair samples may only work with mtdna

26 Dog DNA 38 Autosomes and XY sex chromosomes Cats are usually witnesses Dogs are more often perpetrators Discrimination from STRs is very high Higher than for cats Not as high as humans Again, depends on the breed of dog Hair samples may only work with mtdna

27 Species Identification DNA testing is very useful for identifying the species of an animal Or more often of some meat In cases of poaching and bushmeat mtdna cytochrome b gene is very diversified in different organisms Easy way to identify organisms genotype cytochrome b

28 Plant DNA Two primary uses for Plant DNA testing: 1. Identifying the scene of a crime Suspect may be linked to a scene Link victim to suspect s surroundings Evidence that victim s body was moved 2. Forensic Drug investigation Identifying source of illegal plants Especially Marijuana plants

29 Interaction with plants Suspect may pick up plant DNA at crime scene to connect him Suspect may leave plant DNA brought with him from his home/work Victim s body may carry plant DNA to prove that body has been moved Genetic testing may identify a specific species of plant Or may identify the exact plant/tree

30 Illegal Drugs DNA testing DNA testing may link: Grower with product Individual with product Track distribution over time/space Marijuana most common in US Poppies and other plants also If plants are propagated clonally cannot tell individual plants apart Identical DNA

31 Plant DNA testing Plant s DNA Profile three methods: 1. Randomly amplified polymorphic DNA markers (RAPD) Primers work with any plant species 2. Amplified Fragment Length Polymorphisms (AFLP) Like RFLPs only more specific 3. STRs

32 Insect DNA Insect DNA may be used: Link suspect to crime scene Or to victim Prove that victim s body was moved Especially with rare insects Or if moved far distance Calculate time of death of victim s body Based on types of organisms and what stage of growth they are in

33 Microbial DNA Microbial DNA: Calculate time of death Based on types of organisms and what stage of growth they are in Aquatic microbes provide evidence of fresh or salt water involvement If body is found on dry land evidence of drowning or that body was moved Bioweapons

34 Challenges of Non-Human DNA When presenting the evidence in court juries are much less likely to understand complexities Further challenges: Novelty of application Validity of scientific methodology Validity of statistical calculations Lack of reference databases Lack of experts to testify in court

35 Any Questions? Review Chapters me at least 2 questions you have about these chapters Next class will be review for Exam Exam Two February 23 rd

36 Obtaining sequence Sequence in both forward and reverse directions Or else sequence strands in duplicate Goal is to always have at least 2X coverage of any stretch Sequence is determined with: Two independent forensic analysts examine all sequences to be sure they agree with sequencer s calls Program that automatically aligns pieces

37 Reporting sequence Nucleotides letters: N = unknown base R = A or G (Only know it s a Purine) Y = C or T (a Pyrimidine) Insertion =.#letter (# of bases) Example.1C Deletion = dash or D, d or del Example 390- or 309D

38 Sequence Quality In order to trust mtdna profile the sequence must be high quality Possible errors: Mistakes in reading sequence Sample mix up Contamination Using wrong nomenclature for profile Alleles cannot be compared between labs