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.

Gel electrophoresis is a gel technique that separates DNA and proteins based on their mass, by means of an applied electrical field that passes through an agarose or polyacrylamide gel. The concentration of agarose in the gel is commonly 0.8% to 1.0%, since agarose is expensive. The gel is embedded on a buffer, pH of 8.3, which keeps the pH of the solution at equilibrium. Assuming that at typical pH, DNA is negatively charged, denatured protein samples are placed in the wells located on the negative electrode side; hence the positive electrode side is located at the other end of the gel. As the positive and negative electrode sides are connected to a power source, protein sample migrates to the positive side of the gel. Migration of proteins is not necessarily based on their mass or mass to charge ratio; protein migration across the gel is based on their size. In other words smaller molecules will travels further than bigger molecules. Since the SDS gel contains agarose or polyacrilamide, molecules have to be small enough to migrate to the other end of the gel without getting stuck on the way.

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

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

exception of identical twins, DNA fingerprinting is a reliable method used to determine the parents of a given baby. DNA fingerprints can be derived from restriction

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).

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

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.

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.

Restriction enzymes are important for many aspects of molecular biology such as cloning and restriction mapping. The enzymes are also useful since there are a lot of restriction enzymes that cut the DNA at different locations. The importance of different enzymes cutting different sites makes it possible for scientists to carry out restriction mapping and ultimately know the length of the DNA and locations where restriction enzymes cut on the

DNA fingerprinting, also known as DNA profiling, is a technique used in forensic science that identifies individuals based on various characteristics of their DNA. Although the DNA sequences between humans are 99.9% identical, DNA fingerprinting is able to distinguish between individuals due to the presence of specific sequences within the non-coding region of the genome known as satellite DNA. This satellite DNA consists of long stretches of DNA made up of repeating base sequences known as short tandem repeats (STR). These STRs considerably vary in length between individuals, particularly between unrelated individuals, allowing exact individuals to be identified. One major use of DNA fingerprinting is in

Agarose is a linear polysaccharide unit made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is one of the components of agar that is a mixture of polysaccharides isolated from certain seaweeds. Agarose is usually used in concentration between 1% and 3%. The pore size in the gel is controlled by the initial concentration of agarose; large pore sizes are formed from low concentrations and smaller pore sizes are formed from the higher concentratins. [1][2][5]

This technique simultaneously detects lots of minisatellites in the genome to produce a pattern unique to an individual, or a DNA fingerprint.

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:

Agarose is a linear polysaccharide made up of basic repetitive units of agaribose, which comprises the alternating units of the galactose and 3,6-anhydro galactose. It is approximately at the concentration between 1-3%. Agarose gels are prepared from suspending the dry agarose in the aqueous buffer, then boiling the mixture until a clear solution is formed. This is poured and allowed to cool down to room temperature to form a rigid gel. Agarose gel is formed by the polymerization of D-galactose and 3,6- anhydro –L-galactose. It is