1. DNA fingerprinting

2. DNA fingerprinting is used to determine paternity Look at the DNA of the mother, father and child Could these parents produce this child?

3. DNA fingerprinting 2 methods Southern Blot and Restriction fragment length polymorphism PCR and VNTRs

4. Different types of DNA We have talked about plasmid DNA In DNA fingerprinting you are looking at genomic chromosomal DNA –You are looking at a persons entire genome which is very large –We will do some tests to look at smaller pieces –Remember that at each locus, half of your DNA comes from your mother and half from your father

5. DNA fingerprinting by Southern Blot Southern blot and RFLPs (restriction fragment length polymorphisms) RFLPs – variation in length of genomic segment between 2 restriction enzyme sites Use probes corresponding to the RFLPs Some people may not have these restriction sites

6. DNA fingerprinting by PCR DNA is analyzed for specific variable number of tandem repeats throughout the genome (VNTRs) –Run of repeated nucleotides (4-40) Each individual will usually inherit a different variant of each VNTR from their mother and from their father 2 unrelated individuals will therefore NOT contain the same pair of sequences PCR – primers on either side of the VNTR

7. Other methods STR = short tandem repeats, 4-5 repeats, examined by PCR Mitochondrial DNA is maternally inherited –Determine if children have the same mother Y chromosome analysis –Determine if male children are related These are different approaches but we will not study them in this lab

8. Uses of DNA fingerprinting Identification of a criminal Exoneration of a person for a crime Paternity testing Identification of human remains

9. Question? Why can blood typing only exclude a suspect but DNA fingerprinting can provide positive identification with relatively great accuracy?

10. Southern Blot

11. Analysis of RFLPs by Southern Blot DNA extraction Digestion with restriction enzymes Electrophoresis Southern Blot and hybridization with radioactive probe Detection of RFLPs by autoradiography Reprobe Southern Blot with additional probes

12. DNA extraction DNA can be obtained from many different sources –Blood –Semen –Hair –Saliva –Tissue from a dead person

13. How do you determine who committed the crime? Get DNA from the crime scene from one of the different sources Get DNA from the suspects –Usually from their blood but can use other sources Compare the results

14. Cut DNA with restriction enzymes Restriction enzymes will cut out the regions with the repeated segments but their length will be different in different individuals These are the regions that are specific to each person These are the regions of DNA that will be compared between the crime scene DNA and the suspects DNA

15. Cut chromosomal DNA with restriction enzymes You will get a large number of different size fragments You will get a smear on a gel

16. Electrophoresis and Southern Blot Electrophoresis separates fragments based on size DNA in the gel is denatured by soaking in NaOH –Why do we want to denature the DNA? Single stranded DNA is transferred to a membrane Radioactive probe added Look for regions of hybridization – restriction fragment length polymorphisms (RFLPs) Detected by autoradiography

17. Southern Blot results

19. PCR

20. Use PCR to do fingerprinting need primers to surround the region of the VNTRs

21. Fingerpinting by PCR Notice each person has a different number of repeats at a specific locus or specific VNTR These are detected by electrophoresis following PCR Which individual could have left forensic sample F? The more loci used, the more accurate the analysis

22. Advantages/Disadvantages of PCR and Southern Blot PCRSouthern Blot More sensitive – less DNA needed More DNA needed Faster – a few hoursSlower – at least one day Use a single primer at a time Can reprobe with additional probes Less specificMore specific

23. Results from fingerprinting 3 possibilities No match –Suspect excluded from the group who could have contributed the evidence Inconclusive –DNA old, contaminated or not enough sample Match between 2 samples –Suspect included in the group who could be the source of evidence –Not 100% certainty – with more probes, greater certainty