DNA Biology and Evidence

1. DNA Biology and Evidence Harry R Erwin, PhD CIS308 Faculty of Applied Sciences University of Sunderland

2. Topics in DNA Forensics • Introduction • DNA structure and the genome • DNA handling procedures • PCR • The use of short tandem repeat data • DNA statistics and evidence • DNA databases • Other markers

3. References • Goodwin, Linacre, and Hadi (2007) An Introduction to Forensic Genetics, Wiley. • Butler (2005) Forensic DNA Typing, 2nd edition, Elsevier. • Jablonka, & Lamb. (2005). Evolution in Four Dimensions, MIT Press. • Alberts, B., A. Johnson, et al. (2008). Molecular biology of the cell. New York, Garland Pub. • Erwin, H. R. (2005). Symbolic and Episymbolic Evolution During Cultural Transitions. University of Nottingham. • Wikipedia articles on Gene Expression and Neo-Darwinism, accessed 10 January 2010.

4. Lecture Plan • DNA biology • Handling of DNA evidence • Discussion: DNA evidence and privacy

5. Neo-Darwinian Theory • Natural selection. • Variation (mutation) • Followed by selection based on fitness • Fitness. • Differential long-term survival of genes • Inheritance • Phenotype and genotype

6. Types of inheritance • Genetic inheritance • Epigenetic inheritance • Behavioural inheritance • Symbolic inheritance • Episymbolic inheritance

7. Genetic inheritance • Genetic inheritance is based on DNA and “is the fundamental system of information transfer in the biological world”. • Genes in the biological system are not causal; rather they encode components of genetic networks whose development produces the phenotypes and behaviours we observe. • These networks involve hundreds of proteins and are strongly dynamic, even when their dynamics preserve a homeostasis. • This is the primary type of inheritance we’re concerned with. The other four types are mentioned merely for completeness.

8. Epigenetic inheritance • Epigenetic inheritance is similar to genetic inheritance, but involves mechanisms other than DNA. • This form of inheritance is responsible for tissue differentiation and regulatory system/cell structure preservation. • There are multiple mechanisms—long-term maternal effects, DNA methylation and chromatin marking, gene silencing, and inheritance of regulatory mechanisms. • Epigenetic inheritance is sensitive to environmental demands and stresses, and controls the expression of DNA proteins. • This allows the genome to preserve hidden variation until it is needed to respond to environmental change. • Not relevant to forensics.

9. Behavioural inheritance • Behavioural inheritance is through learning, typically but not necessarily by teaching by members of the kin or social group. • It is more common than might be expected, particularly in primates. Known in all mammals (and in animals as primitive as gastropods). • For example, chimpanzees have simple material cultures that are inherited this way, but an even more interesting example is a group of Japanese macaques who have learned to swim and exploit littoral resources on Koshima Island since the 1950s. These macaques began by learning to wash sweet potatoes provided by scientists studying them, but have since developed a new life-style centred on using the sea. These changes have been super-linear, involving complex interacting adaptations. • Not relevant to forensics.

10. Symbolic inheritance • Symbolic inheritance is also through learning, but is at one remove from behaviour—through the inheritance of signs, texts, oral traditions, and myths, that have to be interpreted to produce behaviour. • Simple languages are known in a number of birds and animals, including the screech calls of phyllostomid bats, which appear to be arbitrary with each association with meaning being learned. • Not relevant to forensics

11. Episymbolic inheritance • I go further than Jablonka and Lamb and have split symbolic inheritance into symbolic and episymbolic components (Erwin, 2005). • Symbolic inheritance in this sense refers to the symbols, texts, oral traditions, myths, and similar cultural elements that a society transmits more or less reliably from generation to generation, while episymbolic inheritance refers to how rules are passed along to decode those symbols, oral traditions, and myths to produce social behaviour. • It is possible that the social control mechanisms expressed in episymbolic inheritance lose their effectiveness under serious stress, allowing accumulated variation in the symbols and texts to produce behavioural variation that allows the culture to evolve quickly in response to its social and physical environment. • Not relevant to forensics

12. DNA biology • Central dogma of molecular biology • Eukaryotic chromosome structure • Inheritance • Mutations • Approaches to sequencing DNA

13. Central Dogma of Molecular Biology: “Transcription proceeds unidirectionally from DNA to protein” DNA mRNA Protein

14. DNA Bases and Structure (from Wikipedia)

15. RNA Bases Ribose replaces deoxyribose and uracil replaces thymine. (The difference is thymine is methylated.)

16. RNA Facts • Uracil replaces Thymine in RNA • RNA does not form a double helix • RNA is less stable than DNA. • Proteins are transcribed from messenger RNA (mRNA) in the cytoplasm outside the nucleus. The mRNA is transcribed from DNA in the nucleus. • The translation from RNA to protein uses short tRNA sequences that bind to specific amino acids and match three successive bases in the mRNA

17. Amino Acids • There are hundreds of amino acids, but only 20 or so are found in most proteins. • There are about 63 transfer RNAs corresponding to the possible RNA triplets. (One triplet is a ‘stop codon’, that terminates transcription since no tRNA matches it.) • The amino acid coding does vary slightly from group to group. • Most of the important amino acids are uniquely characterised by the first two RNA bases in the triplet and are insensitive to the third. This means many DNA sequences may code a specific protein, and protein sequence is less informative than DNA sequence. • The third base can be subject to selection despite not being expressed in the protein.

18. DNA Replication • Chromosomes are duplicated during meiosis and mitosis. • The DNA is duplicated by molecular machines in the cell. • The chromosome is split for a short distance, and duplicated in-situ. • The duplication process involves error checking and correction, which makes mutations rare.

19. DNA Repair • DNA can also be repaired—the damaged sequence is trimmed out and replaced by a corrected sequence using the sequence of the other DNA chain in the chromosome. • Repair is reliable but not perfectly so. Somatic mutations occur (and are important in cancer). • Errors in repair and replication mean that DNA identification is not completely reliable.

20. Inheritance • Cell division involves DNA replication, followed by the copies of each chromosome being pulled apart into the two new cells. • During production of sex cells, one or the other of each pair of chromosomes is used without duplication when the haploid cells are produced. • Except for the X and Y chromosomes, this produces a mixing of chromosomes. • Chromosome segments may also cross over to the other chromosome in the same pair in this process.

21. Cross Over (Recombination) • Recombination allows mixing of genes on the same chromosome. There are some limitations • Cross-over usually doesn’t usually split genes or areas of the chromosome responsible for controlling a gene. (The resulting DNA sequence is inviable.) • If a part of a chromosome is reversed in a sub-population, recombination inside that segment no longer works for all potential pairings in a population. (This seems to have been important in hominid evolution.) • So recombination is not perfect or exact and usually takes place in ‘junk’ DNA

22. Mutations • Single base changes • Moderately common • About a third of the time not expressed in the protein, and so are silent. • Frequently lethal during development. • Reversals of chromosome segments • Very rare (discuss) • STR count (discuss) changes in junk DNA • Usually +/-1, but can be other values • About 0.5% chance per locus per generation

23. Approaches to Sequencing DNA • Protein sequencing • For short proteins (peptides) can be done directly • DNA to protein is many-to-one; hence ambiguous • Most DNA is not transcribed • Some codes proteins and is transcribed • Some controls protein transcription and is not transcribed. • A lot controls development • A lot is ‘junk’ (meaning we don’t know what it is for). • And a lot of the ‘junk’ DNA is repetitive

24. DNA Sequencing • More direct than proteomics • More complete • Still computationally expensive • Needs ‘amplification’ of the DNA to produce enough for transcription. • The most common approach to amplification is the Polymerase Chain Reaction (PCR)

25. Approaches to DNA Identification • Protein coding regions tend to be stereotyped and under selective pressure. Hence not informative. • Control areas are similarly not informative. • Developmental areas are not understood. • Most ‘junk’ DNA is not informative. • Repetitive ‘junk’ DNA is informative, so most DNA ‘markers’ involve repeat counts.

26. Requirements for Forensic Use • Highly polymorphic • Easy and cheap to characterise • Simple to interpret • Consistent between labs • Not under selective pressure • Low mutation rate

27. Repetitive DNA • Mostly variable number tandem repeats (VNTRs) and short tandem repeats (STRs). • VNTRs used at first, but required lots of good quality DNA. • STRs are not as demanding of quality or quantity and have replaced VNTRs in practice.

28. STRs • A short tandem repeat is a short sequence of DNA that is its own complementary sequence—it binds to itself. • These repeat a number of times between two boundaries (which delineate the ‘marker’) and the repeat count is the marker value. • DNA replication works like a zipper slider, and can easily get misaligned, shortening or lengthening an STR sequence during chromosome duplication. • Changes in marker length are fairly rare (about 0.5%/generation per marker) but very useful. • Aside: currently, 67 markers are used in Y-chromosome analysis, which means the mutation rate is about once every three generations.

29. Points to Remember • Most people have more than one (usually two) STR marker values at a given location. • The collection of STR marker values can identify a person very reliably. • Discuss

30. Discussion • Limitations of DNA evidence analysis • Related suspects • Identical twins • Chimeras • The need for care in analysis • Statistics

31. Handling of DNA evidence • Identification of material • DNA extraction • Quantification of DNA • PCR extraction • DNA profile from PCR products • Analysis • Statistical evaluation • Report

32. Kary Mullis • Nobel Prize winner for invention of PCR • Discuss

33. Handling of Material at Crime Scene • PCR is very sensitive and will amplify any DNA found at the scene. • Hence, take care to avoid contamination! • Laboratory procedures have to be very careful (a common problem with poor labs is sloppy procedures). • An aware criminal can contaminate the crime scene irretrievably. • Discuss.

34. Evidence Collection • Full cover to prevent contamination • Sterile swabs for dry stains and contact marks • Lifting from surface using high quality tape. • Liquid blood collected using sterile syringe, pipette, or storage tube containing anticoagulant. • Clothing important—process at lab.

35. Sexual and Physical Assault • Standard swabs • Fingernail scrapings • Hair combings • Contact marks swabbed

36. Presumptive Testing • Test for blood, semen, and saliva • Skin cells on anything touched • Keep reference samples

37. Discussion: DNA evidence and privacy