DNA fingerprinting

DNA Extracted from Human Hair Follicles gave Inconclusive Fingerprints after using AP-PCR and Gel Electrophoresis

The ability to extract DNA from ancient remains, epithelial cells, hair, muscle tissue, and blood, have opened many doors for the identification of people, pedigree analysis, epidemiology, and taxonomy (Anonymous-1, 2001). In this experiment, a DNA fingerprint was attempted to be made using human hair and the latest DNA technology. This involved DNA extraction, AP-PCR, and gel electrophoresis.

The DNA was first extracted using a hair extraction protocol and then amplified with the advanced techniques of Arbitrarily Primed Polymerase Chain Reaction. AP-PCR mimics the DNA replication process in a vial and can be broken down into three parts; denaturing, annealing and copying (Anonymous-5, 1992). This process involves the use of varying primers that will amplify base sequences, within the DNA, which can be used in gel electrophoresis to result in a DNA fingerprint. All organisms have a unique DNA fingerprint which consists of sequences of base pairs (Betsch, 1994).

After repeated attempts, the unique DNA fingerprint of the hair sample was not determined. Changes made to the protocol for AP-PCR included changing the concentration of dNTPs and MgCl 2 , increasing the amount of DNA and primers, and lowering the annealing temperature of the thermal cycler. However, no visible DNA fingerprint resulted after performing gel electrophoresis.

Figure 1: The first attempt at creating a DNA fingerprint from DNA extracted from hair follicles. The process of AP-PCR was used to amplify the amount of DNA before placing the sample in the wells of the gel. Lambda is the Lambda Hind III DNA marker. Lane 1 is the sample using the DL75'-CAATCGCCGT-3' primer, lane 2 used the DL85'-CAGCACCCAC-3' primer, lane 3 used the DL95'-TCACCACGGT-3'primer and, lane 4 used the DL105'-GGATATGCCG-3' primer. The DNA fingerprint was inconclusive however a faint trail in lanes 2-4 can be detected. This may be DNA, but more likely RNA.

The methods of DNA hair extraction, AP-PCR and gel electrophoresis were all utilized to try to create a DNA fingerprint. After extracting the DNA, the process of DNA quantification was very hard to calculate. Following the third DNA extraction, the absorbance of the DNA solution was calculated to be 1.46 which was close to the range determined for the presence of DNA. This means that DNA was possibly present after the extraction but there was not definite answer due to other factors such as proteins.

AP-PCR was therefore run most of the time by estimating the concentration of DNA present after the extraction. This is a possible reason for many calculation errors in the concentration of the components of the PCR mixture. However, for the first PCR preformed, the amount of dNTPs and MgCl 2 was miscalculated. This was fixed in the second PCR performed. Also, for the second PCR, two primers were used instead of one in hopes of amplifying more of the DNA sequences. This would in turn help to brighten the bands that were found from the first gel run as shown in figure 1.

The second gel yielded no results which led to signs that the estimated DNA concentration was incorrect. Therefore, the amount of DNA used for each PCR reaction on the third trial was almost doubled that of the first and second trial. Also, the temperature in the thermal cycler that corresponded to the annealing temperature was lowered to 33.3 degrees C. A positive control of B-subtilis DNA was run along with the hair samples during PCR and gel electrophoresis in hopes of troubleshooting the methods and protocols being used. Since the control did not show up in the DNA fingerprint as shown in lane 5 of figure 4, this could possibly lead to the conclusion that the process of PCR is the problem of the method. However, it does not give us a definite answer that there was DNA extracted from the hair follicles in the first place.

If more time was available, further changes in the PCR concentrations would need to be tried in order to optimize the conditions. This would include increasing the amount of MgCl 2 since it will affect the possibility of the Taq polymerase to work. Also, increasing the number of different primers used during the entire experiment because it is possible that the primers did not bind to any of the sequences of the DNA extracted. If this occurred, there would have been no DNA amplification resulting in no visible DNA fingerprint when performing gel electrophoresis. It is extremely important to have the optimal primer sequences and concentrations in order to have “maximal specificity and efficiency in PCR” ( Löffert et al., 1997).

In addition, because hair is made up of proteins, such as keratin, this could largely affect and inhibit PCR. It is important to not have a large amount of cell debris and protein, from the DNA extraction process, otherwise, PCR could be inhibited (Fernando et al., 2002). Another problem is that hair does not contain a significant amount of DNA when compared to other tissues such as blood. Also, any types of hair treatments could affect the amplification of DNA (Gilbert et al., Unknown). These factors may have all led to the inconclusive results of the DNA fingerprints.