D3S1358 Analysis

In order to determine my genotype, DNA was extracted from the cheek (buccal) cells. After placing the extracted DNA into the Nano drop, the concentration of the sample was observed in order to determine if it would be successful in the polymerase chain reaction. The sample was then placed into a spectrometer to quantify the amount and purity.

Agarose Gel Electrophoresis refers to the movement of a charged particle in an electrical field, usually performed for analytical purposes, and is routinely used for the preparation and analysis of proteins, and DNA – often after amplification of DNA. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. DNA is negatively charged and when placed in an electrical field, DNA will migrate towards the positive pole (anode). An agarose gel is used to slow the movement of DNA and thus separates the size of molecules based on their rate of movement through the gel under the influence of an electrical field.

The typical agarose concentration used for gels is 1%. 3b. If 10X TBE was not added to the gel or running buffer the enzymes would be inactive. TBE buffer makes up an ionic solution which allows current to pass though water. The Tris in the TBE is a basic buffer that keeps the DNA deprotonated during electrophoresis.

tube was set up by adding 15.0ul of the extracted DNA and 10.0ul of the reaction muxture. The

After that, 0.1 ml of solution in Tube 4 was poured into plate 1A, the solution was spread over the surface evenly. These steps were repeated using 1ml from Tube 5 - plate 1B, 0.1 ml from Tube 5- plate 2A, 1 ml from tube 6 - plate 2B, 0.1ml from tube 6 - plate 3A, from tube 7 - plate 3B.

To prepare samples was necesary to place the agarose in a water bath, until it was in a liquid state. At the same time, the granulose cells were ajusted to a concentation of 10^6 cells/ml using PMI Media 1640 (Life Techologies; Carlsbad, CA, USA). A volumen of 25 microlitres of diluted granulose cells was passed to the Eppedorf tube with the agarose at 37ºC, then it was mixed gently.

Thus the enzyme in second tube was diluted to 2.5 U/g DNA. Serial dilution was continued till the sixth tube while the seventh tube was kept without enzyme and served as control. All tubes were incubated for 60 minutes at 37C and the reaction was stopped by adding the sample loading dye (Appendix). The digestion of the chromosomal DNA was observed by agarose gel electrophoresis. An appropriate enzyme concentration was used to digest DNA in the 2-10 kb range.

Gel purification allows us to isolate and purify DNA fragments based on size. The DNA bands from the agarose gel are cut out and purified using some procedure. The band was extracted by using the QIAgen gel extraction protocol. The DNA bands after purification for CD01 and CD02 were shown in Figure 4.3. This steps also considered to be vital components in molecular biology technique as it can help determine the success or failure of the downstream

In this lab, evidence DNA and the DNA samples of two different suspects were available. Two restriction enzyme digests were conducted to cut the samples into smaller pieces. An increase in the number of restriction enzyme digests increases accuracy and decreases the likelihood of false positives. This is crucial in forensic DNA analysis. Finally, the DNA was separated using Electrophoresis. Agarose Gel Electrophoresis is the most effective way of separating DNA fragments of varying sizes and charge (Lee, 2012). Agarose gel is ideal since during gelation, the agarose molecules are held together by hydrogen bonds which form pores uniformly throughout the gel giving it its molecular sieving properties (Lee, 2012).The DNA to be separated is inserted into the pre-cast depressions/wells in the gel (Lee, 2012). The gel is then inserted into a chamber and an electrically conductive buffer solution is added until the gel is completely submerged (Lee, 2012). Positive and negative electrodes are connected to each end of the chamber and an electric current is applied (Lee, 2012). Since the phosphate backbone of the DNA molecule is negatively charged, the DNA will be repelled from the negative electrode and will move toward the positive electrode (Lee, 2012). DNA bands will be visible in the gel with varying distances (Lee, 2012). Smaller fragments will move farther throughout the porous gel.

When the mixtures ready and added to Applied Biosystems wells we have run them to PCR machine as these conditions activation at 50 °C for 2 minutes and at 95°C for 2 minutes and Amplification at 95 °C for 1 seconds; 60 °C for 30 seconds × 40 (Applied Biosystem, 2010).

BamHI and HindIII are the unknown restriction enzymes in this experiment that need to be recognized by agarose gel electrophoresis. Both enzymes have an optimal reaction temperature at 37degrees celcius. BamHI occurs at 5 locations and HindIII occurs at 7 locations. These numbers are important for recognition on the agarose gel for identifying which restriction enzyme is in each reaction.

Optimization following primer design. Once primers have been designed, the assay must be optimized including similar melting temperatures for all of the primers to allow the primers to be run in one thermal cycling protocol resulting in adequate amplification of all amplicons. Since a good indication of success of sequencing reactions is the quality of the PCR template, data collected in the validation process from argarose gels can be used to determine the percentage of time that an amplicon amplifies without problems as illustrated in the example in Figure 11.

There are many different ways that one can extract DNA from biological evidence. Forms of biological evidence are hair cells, tissues, blood, semen, skin cells, muscles, brain cells, bone, hair, waste, and finger nails. Each involves similar but different extraction techniques. Extraction is a technique in which out of the entire sample, DNA is the only thing left to have ("DNA Extraction”). There are four different steps when it comes to extracting the DNA, preparing the sample, binding the DNA to the membrane, wash, and elution of pure DNA (Kennedy). Preparing the sample involves, taking the sample and putting into a microcentrifuge tube and adding buffers into the sample. Binding the DNA to the membrane is important because when the DNA sample is transferred over to the column there is silica (Kennedy). In the binding stage, ethanol is added which allows for the nucleic acids to stick to the silica more, allowing the DNA to bind. The next stage is the wash stage, the wash stage uses a lot of buffers

Figure 3: Pre-Sequencing Sample Preparation. Samples were prepared and amplified on a Biomek 4000 before sequencing

Agarose gel electrophoresis used for the separation of the fragments was based on the migration of the negatively charged DNA to the positively charged anode. DNA has a uniform mass/charge ratio therefore allowing the molecules to be separated by size within an agarose gel such that the distance travelled is inversely proportional to its molecular weight. The rate of migration is controlled by the following; size of DNA molecule, agarose concentration, DNA conformation, voltage applied presence of ethidium bromide, type of agarose and electrophoresis buffer.