Abstract
“Every contact leaves a tract” the locards exchange principle. In most physical crime scenes there is some source of biological evidence found. Almost any intact evidence containing DNA can be extracted and quantitated. DNA extraction is when the nucleic acid from the sample is removed with minimal disruption or degradation. The goal is to obtain the maximum amount of DNA yield in the purest quality. Quantification of DNA helps generate DNA profiles and it helps determine if the DNA is testable. The extractions for this lab were performed by FTA Punch purification, Chelex and Qiagen. The samples used in FTA punch purification were of our own blood obtained through pricking the index finger. Buccal swabs were taken for Chelex and evidence was given for extraction of Qiagen. The quantitation methods performed were Gel electrophoresis, Spectrometry and Real Time PCR. The following lab, Gel Electrophoresis was performed for the Qiagen samples to test for quality and quantity of DNA. UV spectrometry and Real time PCR were performed afterward to quantify and amplify the DNA molecule.
Keywords: Locards exchange principle, FTA Punch Purification, Chelex, Qiagen, Buccal, Gel Electrophoresis, UV Spectrometry, and real time PCR.
Introduction
Forensic DNA technology is in a revolutionizing the way law enforcers investigate crime scenes, DNA is located every living thing. In a Crime scene DNA evidence is very valuable, because its order is very specific and unique. In a scenario when DNA needed to be tested it must be extracted and quantified first before its profile is read. The following extraction methods were performed in this Study, FTA punch purification, Chelex and Qiagen. The purpose of these extraction methods was to obtain the maximum DNA and purest amount for amplification. After Extraction, quantitation was done by Gel electrophoresis, UV spectrometry and Real time PCR. The purpose of these quantification methods was to determine if the DNA extracted was suitable for further testing based on its quantity and quality.
The FTA Punch purification was developed in the 1980’s by lee Burgoyne (3). It can be used on biological samples such as saliva and blood. In this lab, blood samples were taken. The paper is made out of a cellulose based matrix filter paper which contains chemical that protect DNA from degradation and bacterial growth (1). Once a DNA sample is set on the paper it lyses the cells and makes the DNA bind to it. Then a solvent, FTA buffer washes it that is a detergent that washed away all the heme and cellular material yet still keeping the DNA bound to the punch card. The FTA buffer also washed away all the PCR inhibitor. The red color of the bloodstain also washes away. FTA punch purification doesn’t need to be quantified because its results are always consistent.
The Chelex Extraction method was presented in 1991 to the forensic science public (3) , it uses chelating Stryrene divinylbenzne copolymers containing paired iminodiacetate ions directly to the sample (1). The resin chelates the heavy metal ions such as magnesium, which inactivates nuclease activity. The magnesium ions are drawn into the suspended sample and drawn up removing all the magnesium from the sample. Thus removing most of the DNA destroying enzymes and protecting the DNA. The boiling water bath breaks open the cell membranes, denatures proteins and releases DNA as well as denaturing itThe Chelex and cellular debris is located on the bottom and the supernatant containing the DNA is on the top. Chelex extraction is effective for RFLP because Chelex extraction denatures double stranded DNA and it yields single stranded DNA, so Chelex cant be used in spectrometry or gel electrophoresis, only PCR based methods are viable.
Qiagen or QIamp extraction is an method that gives High-throughput DNA extractions. Qiagen is usually the preferred method of extraction because it removes PCR inhibitors more efficiently than other methods and its high DNA yields. In Qiagen extraction the Nucleic acids are selectively absorb to the silica columns due to the presence of chaotropic salts such as guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, and sodium perchlorate (3). The choatropic salts disrupts the Hydrogen bonds and denatures the proteins as well as making the nucleic acids more stable (1). The solution needs to be more acidic than 7.5 for the DNA to be absorbed to the silica more efficiently and to wash away the impurities more proficiently.
Gel electrophoresis in a forensic setting is a quantitation method that is used to separate fragments of DNA by their size. The separation patterns in DNA is unique in in each person, in a crime scene is a forensic scientist can perform gel electrophoresis to match the suspects DNA from the crime scenes DNA. The separation of the DNA fragments are cause by an electrical field applied to an gel, which causes migration at a certain rate, along with distance travel is all dependent on the size of the DNA fragments and the concentration of the gel (2). The gel is composed of aragose gel, derived from seaweed, it contains pores that the DNA can travel in and is easily prepared. The lesser concentration of aragose yields better resolution of smaller fragments and a higher concentration yields better separation of the larger fragments in the sample. Increasing the voltage increases the separation speed. In DNA, the Phosphate groups give up their H+ ions making the DNA fragments negatively charged (2). When an electric field is introduced the DNA fragments will migrate away from the cathode and move toward the positive charge, the anode. The electrical field makes heat, too much heat can cause the gel to melt. Two buffers are used in electrophoresis Tris-acetate-EDTA (TAE) and tris-borate-EDTA (TBE), two dyes are also used for loading and tracking Xylene cyanol and bromophenol blue. The Gel is stained with Ethidum Bromide, which is a intercalating dye that can move into the grooves of the double helix and be bound to it however it does not intercalate with RNA since it is single stranded and has no double helix. Ethidum bromide fluoresces in UV light. After it is scan and photographed the gel provide the information to calculate the total yield of all DNA and its pattern.
In UV spectrometry, nucleic acid absorbs UV light. Nucleic acids absorb ultraviolet light at 260 nm. The spectrometer measures the light that is passed through the sample. The more light the sample absorbs the more purer the sample is. The sample may be contaminated so it affects the purity, which affects the uv absorbance in the sample. To determine purity of the sample the 260/280 ratios is used, 260 nm measures for nitrogenous bases from RNA and DNA and 280 measures for purines and aromatic rings from proteins. 1.8-2.0 is optimal RNA purity and 1.65-1.85 is optimal DNA purity anything less than 1.65 concludes there might be to much protein contamination. The spectrometer used was a Nanodrop UV spectrometer; it requires only a small amount of DNA sample.
Real time PCR is an method that was introduced by Hihuchi at the Cetus corporation in the early 1990’s, it analyzes in cycles the change in fluorescence signal that results in amplification of a target sequence during PCR (4). Real time PCR can be performed with degraded DNA and low quantity of DNA. In this lab, the use of a intercalating dye was used, SYBR Green which had a high affinity for double strand DNA. The assay can detect the formation of any PCR Product. There are three phases in the PCR, exponential amplification, linear amplification, and the plateau region. The best phase to measure fluorescence is the exponential phase, because the amount of product and input DNA is more consistent in that phase.
The three extraction methods from this study showed that FTA paper purification can extract liquid DNA samples such as blood or saliva, giving consistent and doesn’t need to be quantified. Chelex extraction showed that it doesn’t work well with Gel Electrophoresis because it is single stranded and it also isn’t clean as qiagen, because of the result obtained in UV spectrometry showing that none of chelex sample was close to 1.65-1.85 when measuring for purity. Qiagen extraction showed to be the best method of all because it can be used in all three quantification methods.
