Group 5_ Luminol & DNA

1. Influence of the Luminol Chemiluminescence Reaction on the Confirmatory Tests for the Detection & Characterization of Bloodstains in Forensic Analysis Julio Abreu, William Chauvin, Conor McMenamin, John Vitale, Andrew YoungstromBIOL 1115Dr. Missy McElligot16 February 2012

2. Introduction • Detection of crime scenes • Focus police work • Luminol • Detection of invisible blood stains • Evaluation of whether or not luminol negatively affects blood and DNA testing

3. Objectives • Verify effect luminol has on blood and DNA testing • Evaluate dependence on incubation time • Evaluate dependence on luminol concentration

4. Method of use in CSI • Luminol applied to a surface via spray bottle • Detects the presence of minor, unnoticed or hidden bloodstains • Diluted down to a level of 1:106 (1 μL of blood in 1 L of solution) • Used to trace blood trails or splatters • Blood has distinct slow blue glow

5. Luminol dissolved in a base to deprotonate nitrogens • Luminol reacts with an oxidizing agent • Removes N2 • Replaces the Nitrogen with Oxygen • Protons released in the form of blue light (photons) as the molecule falls to the ground state Luminol Reaction

6. Testing • Human gamma-globulines in an immune-hematological assay • Gamma globulines protect against bacterial and viral infectious diseases • Assay measures activity of immune-related parts of blood • X and Y chromosomes quantified using Plexor HY System • Plexor® HY assay - DNA detection down to 6.4pg  • Plexor® HY System determines the concentration of total human DNA and male human DNA simultaneously • Tests for false-negative results that may occur

7. Methods: Investigation 1

8. Methods Group 1: • 21 samples • 100 µL of blood mixed with 200 µL of luminol solution • Luminol concentrations of 0.50%, 2.50%, and 5.0% p/v  • Samples incubated and measured at: 1, 4, 6, 8, 12, 24, 48, 72 hours • Immune-hematological assays for gamma-globuline detection performed on the samples • DNA extraction from samples and quantified

9. Methods: Investigation 2

10. Methods  Group 2: • 21 samples • Serial dilution of DNA and luminol solution • 50 to 0.032 ng/µL  • Luminol concentrations of 0.50%, 2.50% and 5.0% p/v  • Incubated for 8 months • Quantitated using real time PCR on a Bio-Rad IQ5 qPCR instrument

11. Results Figure 1.DNA quantitation of the Group 1 samples with luminol                 solutions at varying concentrations. • Figure 1 • Group 1 - varied shorter incubation time • Incubation time and luminol concentration did not affect DNA concentration 

12. Results • Table 1 •  Group 2 - long constant incubation time for all samples • DNA amount after 8 month incubation • DNA can be detected even at low solution concentration levels DNA amount after incubation of 4 µL of DNA and 4 µL of  luminol solution at varying concentrations by eight months.

13. Conclusion • Luminol impacts the identification of the blood splatter • Does not impact DNA identification • After long periods of incubation in the luminol solution, DNA concentration remains unaffected • Important because luminol should not affect or otherwise impact the identification of DNA profile

14. Significance to Biology • Biochemistry • Iron within hemoglobin of red blood cells acts as a catalyst for the luminol solution to begin a redox reaction • Reaction reduces the luminol and produces photons • Emits a blue glow • DNA • Autosomal DNA: all chromosomes that are unrelated to the X,Y sex chromosomes • Makes up genetic "fingerprint"

15. Article Citation Santos, V.R.D., W.X. Paula, and E. Kalapothakis. "Influence of the Luminol Chemiluminescence Reaction on the Confirmatory Tests for the Detection and Characterization of Bloodstains in Forensic Analysis." Forensic Science International (2009): 196-97. Web.

16. Influence of the Luminol Chemiluminescence Reaction on the Confirmatory Tests for the Detection & Characterization of Bloodstains in Forensic Analysis Julio Abreu, William Chauvin, Conor McMenamin, John Vitale, Andrew YoungstromBIOL 1115Dr. Missy McElligot16 February 2012