Dna Fingerprinting Lab Report

Using Gel Electrophoresis and DNA Fingerprinting to analyze DNA samples

Laquandria M. Gibson

April 14, 2017

BSC2010L

Section #22

Sarah Ellmallah

Introduction

All cells contain a complex structure known as deoxyribonucleic acid (DNA). DNA is a chemical that determines how we are. The multiple combinations of its components are what makes a difference in each person. Long molecules of DNA are organized into chromosomes, which are grouped into 23 pairs. Then the chromosomes are broken down into short segments of DNA known as genes.

A gene is a basic physical and functional hereditary unit. Every gene contains a sequence of DNA that occupies a locus on a chromosome (Upadhyaya, 2017). Genes act as instructions to make proteins, varying in

size, giving cells their shape and structure. Genes occur in alleles, which are forms of the same gene with small differences in their sequence of DNA base. Each gene is inherited from each parent. While humans have the same genes that are arranged in the same order, a gene will have 1-3 bases that vary person to person; changing the shape and function of a protein, making it impossible for two people to have the same exact DNA sequence. Another explanation could be contributed to genetic mutations in an individual from radiation or toxic chemicals or an error when DNA is replicated can also lead to DNA not being the exact same pattern (Upadhyaya, 2017).
Although, no two DNA patterns can be an exact replication, mistakes occur with the confusion of two different DNA samples. The most accurate identification of DNA sampling would be DNA fingerprinting, involving the analysis of different sizes by electrophoresis, a technique that utilizes an electric field to separate DNA, RNA, and proteins. As DNA, RNA, and some proteins are negatively charged, there will be a move towards the positively charged pole. And gel electrophoresis includes the movement of charged molecules in a buffer solution (Cassill, 2015). The gel is composed of a buffer solution containing agarose, a polymer that easily forms a gel-like material at room temperature (Cassill, 2015). Even though DNA fingerprinting does not compare all of DNA’s structure, it does compare the different cuts made by restriction enzymes, molecules that attach to DNA at the recognition sites and results in cutting the DNA strands (Upadhyaya, 2017). Common restriction enzymes used in DNA fingerprinting are HIND III and ECOR I and because, as mentioned, all alleles are different in their base sequences, recognition sites for restriction enzyme tend to vary based on the individual (Upadhyaya, 2017).
Materials
Restriction Enzyme Digestion
In order for DNA samples from suspect one and suspect two to be digested by two different restriction enzymes, four reaction tubes were required, labeled 1-4. In each reaction tube, with a micropipette, ten µL of reaction buffer was used. All the samples were prepared based on the given chart (shown below as Table 1) (Upadhyaya, 2017, p. 58). As far as all four having the same enzymes that was the end, so to not cross-contaminate, the micropipettes tips had to be changed each time (Upadhyaya, 2017). The reaction tubes one and three contained 15µL of enzyme 1 and enzyme 2 was added to reaction tubes two and four. Then, reaction tubes one and two were filled with 15µL of suspect one’s DNA and reaction tubes three and four were filled with suspect two’s DNA. Each tube totaling a final volume of 40µL each. After both DNA and enzymes were added to all the tubes, as per Table 1, they were all capped and gently tapped in order for all contents to be at the bottom and properly mixed and then placed in the incubator for 45 minutes at a temperature of 37ºC and then stored in a refrigerator.
Table 1: Summary of Restriction Enzyme Digestion Reactions (Upadhyaya, 2017)
Reaction Tube Reaction Tube DNA 1 DNA 2 Enzyme 1 Enzyme 2 Final Volume
Crime Scene Samples Crime Scene, cut with Enzyme 1 ready for electrophoresis X -- 45*
Crime Scene, cut with Enzyme 2 ready for electrophoresis -- X 45*
Suspect 1 1 10 15 -- 15 -- 40
2 10 15 -- -- 15 40
Suspect 2 3 10 -- 15 15 -- 40
4 10 -- 15 -- 15 40
*10x Gel loading solution has already been added to the crime scene samples (Upadhyaya, 2017).
For crime scenes one and two, they were pre-prepared. Crime scene one had DNA cut with restriction enzyme one and crime scene two had DNA cut with restriction enzyme two, both containing 10x loading gel solution (Upadhyaya, 2017).
Gel Casting and Staining
The gel cast was prepared based on a 50ml 0.8% gel calculation (Upadhyaya, 2017).

Meaning for 50ml, 0.4 grams of agarose was weighed on a scale. And with a 250ml flask, 50ml of 1X TBE buffer was added as well as the 0.4 grams of agarose (Upadhyaya, 2017). Then plastic was placed over the flask and put in the microwave for one minute. After safely retrieving the hot flask from the microwave with a mitten, it was observed to ensure that the agarose was completely dissolved and was placed on a table to cool down to not burn the casting tray; once cooled 2.5µL of ethidium bromide was added to the agarose solution, which was used to illuminate the gel once placed under UV light (Upadhyaya, 2017). Before pouring the solution into the casting tray, a comb was placed on the middle of the edge of the tray to create wells once the gel was solid, allowing for multiple DNA samples to be used at the same time to be compared against each other. After the gel was solidified, the tray was repositioned as the positive pole was closer to the wells which then would not show much results. After adding 250ml of 1X TBE buffer to the tray, an almost equal amount on both sides, submerging the gel, the comb was removed and the wells were present (Upadhyaya,

2017).
Gel Loading
Before retrieving the four reaction tubes that were prepared prior to the gel, labeled 1-4, four additional tubes were already prepared. Two of which contained markers, standard DNA fragments, and the other two contained DNA from the crime scene with enzyme 1 (CS1) in one tube and enzyme 2 (CS2) in the other tube (Upadhyaya, 2017). For the four reaction tubes prior to the gel, 5µL of 10x gel loading solution was added to each (Upadhyaya, 2017). Inside the wells of the gel, 15µL of the DNA samples went as followed from lane 1 to lane 8: marker, CS1, CS2, sample 1, sample 2, sample 3, sample 4, and marker (Upadhyaya, 2017). After all samples were placed in the wells, the cover of the casting tray was placed on top and ran at 150V for approximately 40 minutes, about 2/3 of the gel. Once the samples were 2/3 of the way, the voltages were turned off and the gel was carefully placed under a UV illuminator.
Results

The gel shown above in Image 1, is under a UV illuminator.

As shown all are migrating downward going towards the positive side, as DNA is negative and the separation of bands are also shown clearly in Image 1.

The bands, all except lane 6, which was DNA from Suspect 1 cut with enzyme 1, were visibly shown as lane 6 did not have any bands that were visible to be compared against for crime scenes one or two.

Suspect’s one DNA is in lanes 4 and 5, with two different restriction enzymes in each. Lane 4, DNA with enzyme 1, did have bands that matched lane 2, crime scene 1 with enzyme 1. Lane 5, DNA with enzyme 2, did only had one marking that was the same as lane 2, the two others, did not match, as it was a different enzyme so when compared to lane 3, crime scene 2 with enzyme 2, the bands still did not align correctly, as lane 5 had an additional band than lane 3 did.

Suspect two’s DNA, in lanes 6 and 7 had two different restriction enzymes in each. Lane 6, DNA with enzyme 1, did not have any visible bands, as shown in Image 1. However, lane 7, DNA with enzyme 2, did match the bands present in lane 3, crime scene 2 with enzyme 2, but were a higher placement than lane 3’s bands were. Then, when compared to lane 2, crime scene 1 with enzyme, even though different enzymes, also did not have any bands that aligned

together.
Discussion