DNA Fingerprinting Methods By: Rachiel Foulks

1. DNA Fingerprinting Methods By: Rachiel Foulks

2. DNA • DNA is the coding for all life as we know it! • No two humans have the same DNA • Varying order of base pairs • Human Genome Project • launched in 1990 and completed in 2003 • purpose: Identifying the 20,000-25,000 genes in human DNA • Much is still unknown about its function even after this major research. • Repeating sequences in DNA have been discovered • These are the focus of DNA fingerprinting

3. Methods • Restriction Fragment Length Polymorphisms (RFLP) • Southern Blotting • Polymerase Chain Reaction (PCR) • Amplified Fragment Length Polymorphisms (AFLP)

4. Southern Blotting/Restriction Fragment Length Polymorphisms • Southern Blotting = Edwin M. Southern • basis of RFLP • Step 1: collection of a DNA sample • Step 2: purify DNA sample • Step 3: use restriction enzymes to digest sample • derived from bacterial organisms • cleave double stranded DNA • type II = recognize specific nucleotide sequences and cleave there • EcoRI = 5’-GAATTC-3’ • Fragments of various lengths, or RFLPs, are created by the restriction enzymes

5. RFLP • Polymorphisms = Areas of variation of nucleotide sequence (insertions, deletions, etc.) • Cause fragments of various sizes to be created • Step 4: Fragments in sample are sorted by size by gel electrophoresis • matching fragments band together • Step 5: Denaturing DNA to make single stranded • soak in NaOH solution and add buffer to neutralize

6. RFLP • Step 6: Blotting using an absorbent material • usually nitrocellulose/ nylon is most durable and has propensity to bind at 500 µg per cm • membrane will draw the buffer and DNA solution up by capillary action • dries binding ssDNA/UV light will help the DNA to covalently bond to the membrane • then treat sheet w/ protein and detergent to saturated remainder • Step 7: Hybridization • probe created with known DNA and labeled with 32P • incubate sample with probe and complementary parts of sample will bind to probe, creating hybridized double stranded sections of DNA • DNA fingerprint is created after exposure to x-ray film

8. Polymerase Chain Reaction • Used to amplify or duplicate a particular section of a DNA strand • Created in 1983 by KaryMullis • Uses a sample of DNA (the template to be copied), two DNA primers, a DNA polymerase enzyme, and a supply of all four nucleotide bases for the making of copies • Step 1: DNA sample denatured by heating to 94°C • Step 2: sample cooled down to 54°C and excess of primers added which bind to complementary sections of sample • primers = oligonucleotidestrands made to complement the 3’-ends of the DNA segment to be copied

9. PCR • Step 3: DNA polymerase added and binds to primers • DNA polymerases derived from thermophilic bacteria, like Taq DNA polymerase from • Thermusaquaticus, are best • suited for PCR • Step 4: Sample reheated to about 72°C • so that Taqpol will continue to add bases • New double stranded DNA is created • Several cycles can be done to create as much as needed • Thermal cyclers = make process extremely easy and fast

10. PCR

11. Amplified Fragment Length Polymorphisms (AFLP) • Commonly called AFLP-PCR because PCR is an integral part • Created by Pieter Vosand colleagues in 1995 • Very similar to RFLP but quicker • Can only be used to study dominant genetic markers • Step 1: DNA sample is collected and purified and restriction enzymes and DNA ligases are added • Typically use Msel and EcoRI • Msel = 5’-TTAA-3’ • EcoRI = 5’-GAATTC-3’

12. AFLP • Step 2: Msel and EcoRI adaptors added • adaptors = short sections of double stranded DNA that have 5’-ends that complement the 5’-ends of the Msel and EcoRI fragments, respectively • have a different nucleotide base on the 3’-end than normal • causes Msel and EcoRI recognition sites to be destroyed after ligation • Step 3: PCR is carried out to amplify strands made in step 2 • extremely selective primers are made for this (less fragments will be amplified) • Step 4: One primer is radioactively labeled (typically EcoRI) and high resolution electrophoresis is performed

14. Applications • Forensic Science • Epidemiology and Pathology • comparing tuberculosis strains • study of epidemics and geographical disease • vaccines • PCR: determining the presence of Human Papillomavirus in the body

16. References • Brinton, K., & Lieberman, K. (1994, May). Basics of DNA Fingerprinting. Retrieved November 28, 2009, from http://protist.biology.washington.edu/fingerprint/dnaintro.html • Chial, H. (2008). DNA Fingerprinting Using Amplified Fragment Length Polymorphisms (AFLP). Retrieved November 29, 2009, from http://www.nature.com/scitable/topicpage/DNA-Fingerprinting-Using-Amplified-Fragment-Length-Polymorphisms-39051 • Department of Health and Human Services. (1992). Polymerase Chain Reaction - Xeroxing DNA. Retrieved November 28, 2009, from http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.php • DNA Fingerprinting: How DNA Fingerprinting Works. (n.d.). Retrieved November 28, 2009, from http://dnafingerprinting19.tripod.com/id1.html • Garrett, R. H., & Grisham, C. M. (2010). Biochemistry (4th ed.). Boston: Brooks/Cole Cengage Learning. • Human Genome Project Information. (2009, August 12). Retrieved November 27, 2009, from http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml • Khalsa, G. (n.d.). Southern Blotting. Retrieved November 28, 2009, from http://askabiologist.asu.edu/expstuff/mamajis/southern/southern.html • Lander, E. S. (1989, June 15). [Review of the book DNA Fingerprinting On Trial]. Nature, 501-505. Retrieved from http://www.bioforensics.com/sequential_unmasking/Lander_Nature_1989.pdf • McNabb, S. J., Braden, C. R., & Navin, T. R. (2002, November). DNA Fingerprinting of Mycobacterium tuberculosis: Lessons Learned and Implications for the Future. Retrieved November 30, 2009, from http://www.cdc.gov/ncidod/EID/vol8no11/02-0402.htm • Schiffman, M. H., Bauer, H. M., Lorincz, A. T., Manos, M., Byrne, J. C., Glass, A. G., et al. (1991, March). Comparison of Southern Blot Hybridization and Polymerase Chain. Journal of Clinical Microbiology, 573-577. Retrieved November 30, 2009, from http://jcm.asm.org/cgi/reprint/29/3/573?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=Comparison+of+Southern+Blot+Hybridization+and+Polymerase+Chain&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT • Southern, E. M. (1975). Detection of Specific Sequences Among DNA Fragments. Retrieved November 28, 2009, from http://biology.bard.edu/ferguson/course/bio310/Student_%20Presentations/Southern_1975.pdf • Stöppler, M. C. (n.d.). PCR (Polymerase Chain Reaction). Retrieved November 29, 2009, from http://www.medicinenet.com/pcr_polymerase_chain_reaction/page2.htm • Van Embden, J., Cave, D., Crawford, J., Dale, J., Eisenach, K., Gicquel, B., et al. (1993). Strain Identification of Mycobacterium tuberculosis by DNA Fingerprinting: Recommendations for a Standardized Methodology. Journal of Clinical Microbiology, 31(2), 406-409. Retrieved from http://jcm.asm.org/cgi/reprint/31/2/406