Introduction to DNA EVIDENCE

1. Introduction to DNA EVIDENCE January 24, 2019

2. DNA FUNCTION • DNA encodes instructions for making all the proteins of the body • These proteins determine our heritable traits

3. Use In Forensics Why is DNA so useful? • Nearly unique (only identical twins have identical DNA) • Commonly found at crime scenes Is any other type of evidence as unique as DNA? Fingerprints are more unique – even identical twins are different. What is the benefit of DNA compared to fingerprints? DNA is more easily found at the crime scene, and the evidence is more easily interpretable (remember, even experts sometimes mis-identify fingerprints)

4. Types of DNA Evidence What kinds of evidence contain DNA that is suitable for forensic analysis? Blood and bodily fluids (saliva, semen) are the most common evidence Which component(s) of blood contain DNA? Only white blood cells – red blood cell do not have a nucleus Why would saliva contain DNA? The (human) DNA in saliva comes from white blood cells & cells from inside of cheek. Skin cells and hair roots are also good sources of DNA. There isn’t enough DNA in the hair shaft to make it useful for forensic analysis

5. DNA Structure DNA stands for deoxyribonucleic acid. DNA is composed of two chains of nucleotides that are twisted around each other to form a double helix. Ladder representation Molecular diagram A single nucleotide

6. Nucleotides Each nucleotide consists of three parts: • A phosphate group • A deoxyribose sugar • A nitrogenous base There are four different types of nitrogenous bases, each of which differs slightly: • Adenine (A) • Thymine (T) • Guanine (G) • Cytosine (C)

7. The nucleotides bind together to form a long, stable chain. Two chains of nucleotides are held together by hydrogen bonds between the nitrogenous bases. The bases bond together in specific ways, called complementary base pairing: A binds with T C binds with G

8. The information in DNA is the sequence of bases read down a chain. • Every three letters forms a ‘word’ that specifies a particular amino acid • Proteins are folded chains amino acids • The sequence of DNA specifies the sequence of amino acids within a protein The sequence of bases in human genes is 99.9% identical. The 0.1% that differs between people is responsible for all the heritable differences between people

9. Chromosomes, DNA, Genes What’s the difference?

10. Genes are regions of DNA that encode for a single protein. Chromosomes are strands of DNA that have been very tightly wound up and wrapped around proteins. During most of the cell’s life, the DNA is only loosely folded so it can be used to make proteins. But, before cell division, the DNA is tightly folded into the characteristic “X” shape of chromosomes. Fun fact: Each human cell contains ~3m of DNA

11. DNA collection & handling What are the most common sources of DNA evidence? • Blood and bodily fluids • Tissue samples, esp skin • Hair root sometimes Investigators collect blood or buccal swabs (inner cheek cells) for reference samples. DNA storage • Blood and bodily fluids should be frozen • Tissue samples should be dried, then frozen

12. DNA Extraction Before analysis, DNA must be extracted from cells. • Soaps are used to dissolve the cell and nuclear membrane. • Enzymes are used to break down the proteins that are bound to DNA. • Salt is used to stabilize the DNA • Alcohol is used to precipitate the DNA DNA extraction from banana – remember 9th grade?

13. DNA Amplification Often investigators find very little DNA at a crime scene, so it must be amplified, or copied, before analysis. DNA is amplified using a process called polymerase chain reaction (PCR).

14. PCR Steps 1 • Heat is used to separate the two strands of DNA. • The solution is cooled and primers – short pieces of nucleotides that can begin the replication process -- are added. • Taq polymerase, an enzyme that will attach individual nucleotides to the growing strands is added. • Once the chain is complete, the process is repeated. These reactions repeated dozens of times within a thermal cycler – it repeatedly heats and cools the solution at appropriate intervals to allow the DNA to be copied. The amount of DNA doubles each cycle. In three hours, one million copies can be made. 2 3 4 Watch me!!

15. What are the steps to processing DNA that we have discussed so far? • DNA collection – collecting evidence • DNA extraction – extracting DNA from cells • PCR – amplifying (copying) the DNA Which genes would be amplified? All ?? Nope, only the ones that the investigators are interested in. Investigators can select which genes to copy by adjusting which primers they use. Last step … • Separating and identifying the alleles using capillary gel electrophoresis More on this next class!

16. Gel Electrophoresis Gel electrophoresis is a method of separating molecules (usually proteins or DNA) by size and/or charge. • Samples are loaded into wells (indentations) of an gel • An electric current is put across the gel with the positive electrode on the side opposite the wells • Negatively charged particles (such as DNA and some proteins) diffuse across the gel towards the positive electrode

17. Gel Electrophoresis Watch me! • Dyes are often added to the samples so that you can see them as they move down the gel • Molecules move down gel at different rates due to size and charge • What molecules move fastest? • smaller & more negatively • charged move faster/further • DNA fragments all have the same • (negative) charge. So, DNA • fragments separate purely based • on size. • Scientists will also run known samples for comparison

18. Gel electrophoresis Question A scientist added four substances to this gel. • Small, highly positively charged chemicals • Small, slightly positively charged chemicals • Large, highly positively charged chemicals • Large, slightly positively charged chemicals Which is which?

19. Gel electrophoresis Question 2 The story … Police searched the home a person suspected of murder and found a shirt with blood stains. The blood type of the suspect, the victim, and the blood on the shirt was all O+, so the forensic serologist examined additional blood factors, including PGM.

20. Gel electrophoresis Question 2 • What PGM factor does each sample have? suspect: 1- victim: 1+2- clothing: 1- • Who is the most likely source for the blood stain? suspect • What is the purposeof a reference sample? allows you to match bands to known types

21. Capillary Gel Electrophoresis • The gels shown in the previous example are ‘slab’ gels. • Most of the time, forensic DNA analysis is done using a newer technique called capillary gel electrophoresis. • Capillary gel electrophoresis is useful because it is better at separating pieces of DNA with small differences in size AND is easily automated.

22. Capillary Gel Electrophoresis Watch me! • DNA fragments are fluorescently labeled and are separated along a thin glass capillary tube (rather than an gel slab). • A detector ‘reads’ the fluorescently marked fragments as they pass through the column, and showing peaks for each fragment size. • Samples of known size are also run through the column for comparison.