Length Polymorphisms

Restriction enzymes are a tool that allows us to pinpoint human identity down to single differences in our DNA. Work through the following simulation so you can see these molecular scissors in action.

The idea behind Gel Electrophoresis is that we inject a slab of gel with the DNA we found at the crime scene. We then inject the same gel, next to the crime scene DNA, with suspect 1’s DNA and suspect 2’s DNA. We then send an electric current through the gel and wait for the results. The smaller molecules in the DNA will travel farther than the bigger molecules because the bigger ones will have difficulty making its way through the microscopic beads in the gel. After the separate bands appear in the gel, we stain it with a special chemical called Ethidium Bromide to give it a color under the blue

Gel electrophoresis is a method of taking DNA samples and turning them into visuals that can be compared and analyzed. First, DNA fragments are placed on a layer of agarose gel, and when electricity is added, the DNA fragments move through the gel. The smaller fragments move faster than the larger ones. Afterward, you can see how the different lengths of

I added 1 g of the agarose powder to 100 ml of TAE buffer in an Erlenmeyer flask. Using a magnetic stir bar, the agarose is heated until it is a completely clear solution. The agarose is poured into the chamber to make wells deep enough to hold 30 µl of solution. I inserted the comb in the chamber and waited for the gel to harden. To prepare for the samples, 10 µl of the XC loading buffer was added into the PCR tube. The professor demonstrated the techniques to load the gel with the DNA samples. After learning the techniques, I added my PCR sample in one the the wells. To run the gel, the power supply was set to 100V for approximately 30 minutes. To stain the gel, the gel was removed from the electrophoresis chamber and was placed into the FastBlast staining tray. The gel was placed in the staining tray for 2 minutes. Then the gel was rinsed for 10 seconds in Wash Tray 1. After rinsing for 10 seconds, the gel was washed for 5 minutes in Wash Tray 2. After agitating the gel for every minute in Wash Tray 2, the gel was washed for another 5 minutes in Wash Tray

The results for this particular lab were accurate; however, inaccuracies could arise from contamination of the PCR reagents, a degraded DNA template, incorrect temperatures during PCR, pipetting errors, or mistakes in the preparation of the gel agarose. For instance, if the ethidium bromide were not added to the gel then the results would not be visible under UV light. If the DNA template were degraded then the DNA would not be amplified during PCR, thus there would be no results to observe on the gel. Contaminated PCR

(n.d.). Agarose gel electrophoresis. Retrieved on December 6, 2014, from http://faculty.plattsburgh. edu/donald.slish/Electrophoresis.html. 5.

Gel electrophoresis is another common technique in molecular biology that separates DNA fragments based on size using an electrode and agarose gel (Aaij et al., 1972). The negatively-charged DNA fragments move along the gel from the negatively charged electrode to positively charged electrode, while also moving through the small pores in the agarose gel, thereby separating the DNA fragments based on size and charge. The more negatively charged and smaller a molecule is, the farther it will travel along the electrode. The distances of the fragments are compared using the dark bands that appear in the gel following the procedure (Aaij et al., 1972).

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.

Sir Alec Jefferys discovered DNA fingerprinting in 1984 after he realized that variations in human DNA could be detected in the form of short sequences of repetitive DNA (4). On average, 99.9% of human DNA is the same from person to person however; the 1% remaining is what makes each person unique. Although this seems like a small amount, there are approximately 3 million base pairs in these short, repetitive sequences that distinguish one person from another.

The following figure shows the agarose gel results of the digested DNA plasmids 1 and 2 as well as the molecular weight markers. This allows for the visualisation of DNA by adding fluorescent dye SYBR Green to the gel loading dye which was then added to the restriction digest. These results belong to Jessica and Vince in Group 13. My results did not provide clear bands above the top maker, which may be a result of the restriction digest not working properly. However, these results below illustrate two clear bands of each of the digested plasmids and the bands are below the top maker.

To prepare the 2% agarose gel, 1.0 grams of agarose and 50 ml of 1X TBE electrophoresis buffer were mixed in a 125 ml glass flask. The mixture was heated in the microwave for 20 second cycles until the agarose was dissolve. The agarose was set aside for 10-15 minutes to cool down. 2.5 µl of ethidium bromide was added to the agarose mixture to stain the DNA and allow the banding patterns to be visualized. Finally, the solution was poured into the gel casting tray and the comb was inserted at the top. The gel was set aside to cool down until

Since the charge per unit length (owing to the phosphate groups) in any given fragment of DNA is the same, all DNA samples should move towards the anode with the same mobility under an applied electrical field. However, separation in agarose gels is achieved because of resistance to their movement caused by the gel matrix. The largest molecules will have the most difficulty passing through the gel pores (very large molecules may even be blocked

Presumably primer sites are randomly distributed along the target genome, and flank both conserved and highly variable regions. Wide variation in band intensity can be shown to be reproducible between experiments, which could be the result of multiple copies of the amplified regions in the template or the efficiency with which particular regions are amplified (Bardakci, 2001). The polymorphic bands obtained from a RAPD can also be cloned for further analysis. The major advantage of RAPD includes that, it does not require pre-sequencing of DNA (Nandani and Thakur, 2014). RAPD-PCR fingerprints have been successfully used in defining genetic diversity among different species (Lynch and Milligan, 1994). For example, the RAPD method was used to generate specific fingerprint patterns of ten different species: including wild boar, pig, horse, buffalo, beef, venison, dog, cat, rabbit, and kangaroo (Koh et al., 1996). RAPD markers have several obvious features as summarized here as:

Every human being on earth with the exception of identical twins has their own unique sequence of bases in their DNA. With humans DNA being unique just like our fingerprints it can be used to identification of people. This is where the term “DNA fingerprinting” or also “genetic profiling” comes from. DNA fingerprinting just like normal fingerprints can be used to help identify suspects in crimes. The technique is used by forensic scientists.

Due to the DNA’s specificity, samples can be utilised for identification. DNA is a nucleic acid composed of deoxyribose sugar bound to a phosphate group and one of four nitrogenous bases (adenine, guanine, cytosine and thymine). Each section of these three components are referred to as nucleotides, which are joined to the phosphate or sugar of another nucleotide by strong covalent bonds to form a backbone. The nitrogenous bases are joined to complimentary bases of another nucleotide (adenine with thymine, guanine with cytosine) to create a double stranded molecule (Figure 2). To complete the double helical structure, the molecule coils to compact it’s contents. DNA molecules can contain up to two million base pairs, with a human genome containing approximately 3 million base pairs. The random assortment of nitrogenous bases as well as the numerous mutations within certain DNA sequences, results in genetically diverese DNA molecules and genomes between individials.