1. DNA Typing C. Gordeyev (Adapted in part from Larry J. Scheffler, Lincoln High School, 2009) 1

2. Takes advantage of Mini/Microsatellites in DNA DNA that codes for proteins and has important functions is very similar in all humans (conserved) Non-coding DNA is much more variable Microsatellites are regions of tandem repeats of a pattern of 1-6 base pairs (ie - CAGCAGCAG) Minisatellites are repeating patterns of 10-60 bp The number of times that these patterns repeat in a given area of DNA is highly variable in humans http://www.nature.com/nm/journal/v11/n10/fig_tab/nm1005-1035_F2.html

3. Takes advantage of Mini/Microsatellites in DNA An individual will inherit a random half of their mother’s mini/micro satellites and a random half of their father’s (depending on how their chromosomes combine) The chances of 2 individuals (who aren’t identical twins) having the exact same pattern on mini and microsatellites are miniscule –Estimated by some as 0.2536 36 (1 in 5000 billion billion) Therefore each DNA type is unique: a DNA fingerprint

4. Using Restriction Enzymes to study DNA One DNA molecule is far too long to be easily examined We digest (cut up) DNA with restriction enzymes Restriction enzymes will only cleave DNA at a specific sequence http://www-math.mit.edu/~lippert/18.417/lectures/02_PartialDigest/Pictures/100000000000052F000003E8CBEA8CE5.jpg

5. Using Restriction Enzymes to study DNA Each DNA molecule will only be digested at certain locations that have this sequence Two non-identical DNA molecules will have different patterns of where they are digested This produces DNA fragments of different length

6. Gels: Sorting DNA Fragments by Length We load the DNA fragments into wells in one end of a polyagarose gel We run a current through the gel so that the end the DNA is inserted into is negatively charged and the opposite end is positively charged DNA is negatively charged, and migrates through the gel towards the positive end http://img.youtube.com/vi/IWZN_G_pC8U/0.jpg

7. Gels: Sorting DNA Fragments by Length The smaller a DNA fragment is, the easier it is for it to wind its way through the gel to the other end The DNA fragments form a column of DNA Bands: smallest fragments closest to the positively charged end http://www.cbs.dtu.dk/staff/dave/roanoke/fig5_33.jpg

8. BUT If we leave it like this there are so many bands that your column is one big blur We need some way of making this readable and useful We also want to look only at the areas that contain the mini/microsatellites http://journals.prous.com/journals/servlet/xmlxsl/pk_journals.xml_summary_pr?p_Jo urnalId=6&p_RefId=696541&p_IsPs=N

9. SO We transfer the DNA onto a membrane (nylon) and denature it (by use of enzymes) so our DNA is now single-stranded Single-stranded DNA will bind its complement strand very readily

10. Bands light up fluorescently Then we make a piece of single-stranded DNA that’s complimentary to the mini/microsatellite sequence, and attach a fluorescent molecule to it Where this binds to the gel, we see fluorescence This is where the mini/microstellites are http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/05vol31/images/cdr3108_fig3.gif

11. Forensic DNA Analysis When many fluorescent probes are used to create many bands, this is called DNA Fingerprinting When only the 4-5 most indicative probes are used, we call this DNA Typing 11

12. DNA Fingerprinting Process DNA fingerprinting is a multistep process. 12

13. Forensic DNA Analysis Used to identify people in criminal cases.Used to identify people in criminal cases. Used to establish identity, paternity and ancestry.Used to establish identity, paternity and ancestry. Used to study evolutionary changes in speciesUsed to study evolutionary changes in species Used to ascertain genetic disease inheritance (Huntington’s disease)Used to ascertain genetic disease inheritance (Huntington’s disease) 13

14. Forensic DNA Analysis DNA evidence is only as good as the person performing the tests. Care must be taken to guard against contamination for legal evidence to stand.DNA evidence is only as good as the person performing the tests. Care must be taken to guard against contamination for legal evidence to stand. Chain of evidence rulesChain of evidence rules The more individual bands match up, the more valid the resultsThe more individual bands match up, the more valid the results 14