DNA Fingerprinting

Objectives:  To learn the restriction digestion and staining DNA sample.

Introduction: DNA fingerprinting is a very quick way to compare the DNA sequences of any two or more living organisms. DNA fingerprinting can be used to trace the inheritance of genetic disorders, identify origin of a blood or criminal investigation. DNA is a linear, polymeric molecule composed of four different nitrogenous bases: Adenine, cytosine, guanine and thymine. Restriction enzymes are used to cleave the DNA at specific sites based on nucleotide sequences, the common restriction enzymes are EcoR I (GAATTC), Pst I (CTGCAG) and Hind III (AAGCTT). The technique of DNA fingerprinting requires that the DNA be cut up into small fragment using the restriction digestion of DNA samples. DNA is naturally colorless, hard to visible in the gel without UV light. Fast Blast DNA stain can be used as an in-gel stain, by incorporating it into an agarose gel and electrophoresis buffer so that it stains DNA while it is running through the gel. Fast Blast DNA stain molecules strongly bind to the DNA fragments to become visible without the UV light.

Materials: Micropipette and tips, disposable tips cup, 1.5ml microfuge tubes and rack, Lambda DNA stock with labeled S1, S2, S3, S4 and S5, crime scene (CS), EcoR I/Pst I enzyme mix, foam tube holder, water bath incubator, loading dye, 1% agarose, weigh paper, digital analytical balance, microwave, 250ml flask, graduated cylinder, paper towel, TAE buffer, 100X and 500X Fast Blast stain, gel box, gel tray, gel comb, warm water and staining tray.

Methods: Labeled five 1.5ml microfuge tubes with initial and suspect S1, S2, S3, S4 and S5. Used a new micropipette tip for each sample, transferred 10μl of each stock DNA sample from stock S1 to tube S1, stock S2 to tube S2, stock S3 to tube S3, stock S4 to tube S4 and stock S5 to tube S5 labeled. Used a new micropipette tip for each sample, added 10μl of the Enzyme Mix (EcoR I/ Pst I) to each tube and mixed well with pipette. Made sure all the tube caps tighten, put in a foam tube holder and placed in water bath incubator at 37°C for 45 minutes. After incubation, took out and used new micropipette tip for each sample, pipetted 5µl of loading dye to each tube and mixed gently with pipette plunger. Stored in refrigerator at 4°C temperature (Long-term storage should be at -20°C). Run gel electrophoresis to detect the presence of DNA using Fast Blast stain. Weighed out 0.5g of agarose with weigh paper on digital analytical balance, poured into a 250ml flask, used graduated cylinder to measure 50ml of TAE buffer poured in a flask to mix with agarose and covered with paper towel, then put it in microwave, heated about 90 seconds until all agarose dissolved, allowed to cool down between 50°C - 55°C under the hood, added 33µl of 500X Fast Blast to the gel. Set up gel box and the comb in place. Poured the gel slowly into the gel tray, pushed any bubbles away to the side using pipette tip. Let the gel solidified. Removed the comb from the gel and placed gel tray into the gel box, then filled TAE buffer to the gel box until the gel covered with buffer, added 200µl of 500X Fast Blast per 300ml of 1X TAE buffer in gel box. Used a new tip for each sample, loaded 12µl for each sample (CS, S1, S2, S3, S4, S5) into the wells and run gel for 30 minutes. After electrophoresis was completed, turned off the power and removed the gel tray out and carefully slide it into the staining tray to stain the gel with 100X Fast Blast stain for 2 minutes, then transferred the gel into other tray with warm tap water (40° - 55°C). Gently shake the gel in water to wash, repeated to change warm water about 5 times, until the dye no more came out from the gel. Placed the gel on a light background and recorded the results.

Results/Calculations: To prepare to make 50ml of 1 % agarose:
                    1 g   x  50ml = 0.5 g of agarose
                   100ml
Picture: Showed the results between crime scene and suspects DNA fragments in 1% agarose gel with Fast Blast stain.
The well suspect (S3) DNA bands lined up with crime scene (CS) DNA bands, suspect S1, S2, S3, S5 had one band matched to CS. The well suspect S4 was no DNA matched with CS.
             
Conclusion/Discussion: The same restriction enzymes mix (EcoR I/ Pst I) added to Crime Scene and each Suspect DNA samples, DNA fragments were successfully visible in 1% agarose gel with the Fast Blast stain on a light background. The Suspect S3 appeared to be the guilty party, because the DNA bands matched to the Crime Scene. The well S4 was not successfully digestion, may cause by either DNA sample incomplete digestion, DNA sample incubated too long or DNA sample was contamination. Fast Blast DNA stain is nontoxic and noncarcinogenic, but it will stain skin and clothing. Wear gloves and a lab coat whenever handling stain solutions or stained gels.

References:
Advanced Biotechnology Laboratory Manual by BioHealth College, Inc.
http://learn.genetics.utah.edu/content/labs/extraction/