DNA Fingerprinting MethodsBy: Rachiel Foulks

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

Methods

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

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

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

• 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

RFLP

Polymerase Chain Reaction• Used to amplify or duplicate a particular section of a DNA strand• Created in 1983 by Kary Mullis• 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 = oligonucleotide strands made to complement the 3’-ends of the DNA segment to be copied

• Step 3: DNA polymerase added and binds to primers• DNA polymerases derived from thermophilic bacteria, like Taq DNA polymerase from Thermus aquaticus, are best suited for PCR

• Step 4: Sample reheated to about 72°C so that Taq pol 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

PCR

PCR

Amplified Fragment Length Polymorphisms (AFLP)• Commonly called AFLP-PCR

because PCR is an integral part• Created by Pieter Vos and 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’

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

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

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