Identifying an Unknown Tissue Sample to be Agkistrodon contortrix Using Forensic Techniques
Cara (Ginny) Stuart
Diarra Hassell
Bio 2450 Lab 4
Abstract
In modern day science, DNA sequencing plays a huge role in forensics and genetics testing. Scientists are now able to identify an organism from any other if given an adequate amount of ones’ DNA. The purpose of this experiment was to obtain a tissue sample from an unknown organism, and throughout several very strict processes, determine the origin of the tissue. These processes contained procedures that overall allowed the tissue sample to be broken down into DNA segments, and then sequenced using a sequencing machine. After the tissue of the sample was sequenced, it was pasted into an online nucleotide bank that gave the organism’s closest matches. Ultimately, it was determined that the tissue was from Agkistrodon contortrix, more commonly known as the Copperhead snake.
Introduction
DNA is found in every living organism, and contains four nitrogenous bases that can code for genes. Every organism has DNA composed in a genome, which is the complete genetic makeup of the organism. The genome is coded for by the nitrogenous bases, and can distinguish any organism from any other if the base sequence is long enough. Although there are billions of nucleotides (nitrogenous bases) in each genome, the genetic variation of DNA in humans is only 0.1% (Krimsky and Simoncelli, 2011). This is important because in modern day genetics, scientists can now use these vast sequences to look for genes to recognize and find genetic disorders in different organisms. Another application of DNA sequencing is in forensics. Forensics, for example, can be used to amplify STRs (short tandem repeats) using PCR to help create a “DNA Fingerprint” and identify certain individuals (Morton, 1977). This process of identifying the origin of DNA from specimens has made an extreme impact on the law and police forces in cases. Forensic scientists are now able to determine who committed certain crimes, or who was at a scene based on tiny pieces of evidence that contain DNA.
This is relevant to this study because a tissue sample from an unknown origin was obtained, and run through various procedures often used in forensics to ultimately identify the species and genus. In this experiment, a sample tissue of unknown origin was taken and DNA was extracted from the tissue using the most of the Qiagen DNeasy Blood and Tissue Kit and protocol. DNA extraction consists of deconstructing the cell walls, breaking down the proteins in the cell, and then purifying the leftover DNA in the solution (Duff et. al., 2007). This is important because it allows scientists and researchers to extract DNA from organisms for further analysis and ultimately for sequencing.
The next major step in this experiment was Polymerase Chain Reaction (PCR). PCR is an amplification test used to amplify specific DNA sequences (Murray et. al., 2000). Because PCR can amplify the smallest amounts of DNA, it is very crucial that the samples did not obtain any cross contamination, or the wrong DNA could be amplified. In PCR, primers, buffers, enzymes, dNTPs (which are nucleotide precursors), and forward and reverse primers are added into a tube with the template DNA from the DNA extraction. The process of PCR is carried out through a process called thermal cycling, where various cycles of denaturations, primer annealings, and primer extensions occur at different temperatures to allow the various primers, enzymes, and buffers to function optimally. The GeneAmp® PCR System 9700 was used for thermal cycling.
This cycling system allows the DNA to amplify exponentially over the course of many cycles. The success of PCR is determined by running the product on a gel and analyzing the intensity of DNA bands.
After the PCR is proven to be successful using gel electrophoresis, a PCR clean up procedure is conducted to clean the PCR product of excess dNTP’s and primers in the tube. After this is complete, a Cycle Sequencing Reaction (CSR) is conducted.
The Cycle Sequencing Reaction and CSR clean up are used to remove the excess ddNTP’s from the DNA solution and add dye so that a machine can read and sequence the tagged nucleotides (Dong et. al., 2008). Cycle sequencing is very useful in that it allows sequencing of much shorter fragments of DNA than could not be sequenced otherwise (Blazej et. al., 2007). This is especially useful in the experiment that was conducted because there was significant room for error, so the shorter sequences allowed the results to be more accurate with less work.
Finally, the DNA sequences were edited in the GENEIOUS software program and pasted into GenBank’s BLAST® (basic local alignment search tool) to determine the origin of the tissue sample obtained.
Materials and Methods
DNA Extraction
DNA extraction was performed with an unknown tissue sample using the Qiagen DNeasy Blood and Tissue Kit, with an exception in the protocol during steps 17-21. Instead of one elution occurring in these steps of 200L, two were performed with 100L. After the DNA extraction, a gel electrophoresis was conducted using a 1% agarose gel prepared with TBE buffer. Then, 1L of GelRed dye was mixed with 10L of the DNA sample and ran on the gel for 30 minutes at 120V.
Gel Electrophoresis
The DNA bands were imaged using a gel documentation system and then analyzed for success of the extraction. Based on the failed results of the gel electrophoresis, it was determined that a backup sample of DNA be used subsequently in PCR.
Polymerase Chain Reaction (PCR)
The Polymerase Chain Reaction was conducted by adding into a tube, 23.0L of a master mix, made up of 12.0L Green Taq mix and 11L ddH2O. 0.5L of each forward primer (ND4) and reverse primer (Leu) were subsequently added along with 1.0L of the template DNA. A form of PCR called “Touchdown PCR” was used. First, an initial incubation period of 5 minutes was run at 95C for 5 minutes. This was followed by a denaturing step at 95C for 30 seconds, an annealing step at 60C for 1 minute, and an extension phase for 1 minute at 72C. The denaturing, annealing, and extension steps were each repeated for 10 cycles, with the annealing cycle temperature lowered by 1C each cycle. In addition to this, 30 more cycles were conducted with denaturing at 95C for 30 seconds, annealing at 50C for 1 minute, and finally the extension step at 72C for 1 minute. After completed, a final extension period was run for 7 minutes at 72C. Once completed, the reactions were held at at 4C, and then stored at -35C.
Gel Electrophoresis
PCR products were then separated using gel electrophoresis, which was conducted in a %1 agarose gel used with TBE buffer, and performed on 18L of PCR product after staining with 2L GelRed dye. The solution was then loaded into the gel and let run for 30 minutes at 120V, and subsequently examined using a gel documentation imaging system.
Polymerase Chain Reaction Clean Up (cPCR)
PCR clean up was conducted using the ExoSAP protocol by Affymetrix, except in step 1 where the reaction volumes were doubled. Because the gel electrophoresis showed the PCR to be unsuccessful, a backup solution that was successful was used in this step.
Cycle Sequencing Reaction (CSR)
In the cycle sequencing reaction, the first step was to label two tubes, one with “ND4,” and the other with “Leu.” In the “ND4” tube, 6.5L ddH2O, 2L clean PCR product, and 0.5L ND4 were added. Next, 2.0L Big Dye Terminator Cycle Sequencing Buffer was added before storing at -20C until thermal cycling. The above steps were then repeated for the second tube labeled “Leu,” and 0.5L of Leu were added instead of ND4. Thermal cycling was conducted using the GeneAmp PCR System 9700. The procedure consisted of 40 cycles, each cycle containing denaturation for 20 seconds at 96C, primer annealing at 20 seconds at 50C, and primer extension at 60C for 4 minutes. Tubes were then stored at -20C until CSR clean up.
CSR Clean Up
The Centri-Sep by Princeton Separations containing Sephedex columns were used to carry out CSR clean up, and the protocol was followed to yield two electropherograms.
Electropherograms and GenBank
The two electropherogram sequence files obtained from the CSR clean up were uploaded into the GENEIOUS® Basic 9.0.1 software program. The poor quality bases were deleted from each sequence, and then the sequences were aligned. A 50% strict consensus sequence was generated, yielding a new bidirectionally verified sequence that was then pasted into BLAST® to identify the origin of our tissue sample.
Results
The results from the DNA Extraction Gel showed a very faint band, which indicates the extraction was unsuccessful (Figure 1).
The PCR gel results also did not have any bands present. This indicated that the PCR was also unsuccessful (Figure 2).
The final consensus sequence from sample “A44” was analyzed using the GenBank BLAST® program using an 839 base sequence. The BLAST® results showed the highest percent match to the organism Agkistrodon contortrix, or more commonly known as the Copperhead snake, with a 99% match with the DNA in the database. The E-value, or error value, was zero. The accession number was AF156576.1 (Figure 3).
Discussion
In this experiment, a tissue sample was taken from an unknown organism labeled “A44.” The tissue sample was then put through a series of procedures to find its point of origin. The first step in this procedure was to conduct a DNA extraction, which was determined to be unsuccessful by gel electrophoresis. The A44 sample showed non-existent bands in the gel, which means that sufficient DNA was not extracted. If there was a solid band in the gel, this would indicate a positive result, meaning extraction was successful. This failure could be due to the sample tissue of the organism being too large or small, which means the enzymes in the DNA extraction protocol did not function at high efficiency levels. There could also have been the error that the sample was loaded improperly into the gel, which would have also resulted in no bands. Another error that could have occurred is a pipetting error. If any of the enzymes or buffers in the protocol were pipetted wrongly it could have caused an unsuccessful extraction. Because the reaction was unsuccessful, a stock solution already proven to be successful in the gel was used for the rest of the procedures.
In the second part PCR was conducted, and the PCR gel showed no indication of bands for sample A44. A successful PCR would show a solid band of amplified DNA, but instead the sample formed a solid smear. This indicates a failed PCR, which means that the DNA failed to be amplified adequately. A cause for this could likely be an error in pipetting any of the solutions into the tube, but most likely the Green Taq mix. This is because the Taq mix contains dNTP’s, primers, and enzymes among other things and too much of or little of these things could inhibit the PCR from occurring. Too many dNTP’s will inhibit PCR, excess enzyme will cause a smear of the product on the gel, and not enough primer would yield no product, while too much primer would cause primer dimerization (Yang et. Al., 2003). Because the A44 sample showed a smear, it is likely that there was excess enzyme, and that the Taq was pipetted in an incorrect amount.
In the rest of the procedure, the cPCR, CSR, CSR clean up, and sequencing went as planned. The expected results of the final sequences (from the forward and reverse primers) yielded over 700 base pairs each, which was adequate for analysis and editing in the GENEIOUS system, and was able to be used in BLAST® to determine the origin of the tissue without any backups being needed to supply the correct sequences. The E-value, or error value, of the consensus sequence to the sequence in BLAST® was 0. This means that the match to Agkistrodon contortrix is accurate and not by chance. If the consensus sequence of 839 would have been shorter, the E-value could be expected to rise as the accuracy of DNA sequence matches would decrease (Espadaler, 2005). The consensus and organism sequence in BLAST® were matched with a 99% similarity. The accession number of the sequence in BLAST® was AF156576.1. These results indicate a definite positive result in this experiment, and show that the tissue sample was identified correctly. If the sample had not produced an adequate sequence, there would have been some error in any of the above procedures.
In the future, this study could be improved by lessening the confusion of the procedures and protocols. There was confusion during the analysis of the experiment and after among what exact protocols were used in the lab, which makes the errors in the experiment harder to pinpoint. This could be done by writing the steps out in the lab manual in one place without changes, having one document that states all of the correct experimental protocol and procedures, or having every lab/study use the same protocols. This might allow a slightly better understanding of the study overall. The study could also be improved by having more time/practice pipetting small amounts. There were times in class where students were not sure whether their amount was accurately pipetted into the tubes, because some of the amounts were hard to visualize. More practice with smaller amounts using the pipettes, if time given, could potentially lead to more positive experimental results and less stock solution being used.
One extremely important aspect of this study to note is the errors that were made. The PCR and DNA extraction were both unsuccessful. This applies to modern day science and forensics in the way that while in the experiment back up samples were provided, in the real world, there usually would not be backups. This is important because scientists have to use the most accurate protocols for each of these so that the DNA and opportunity of identification does not get lost. This occurs commonly in ancient DNA, which degrades over time. Extremely strict procedures have to be used to carry out PCR, and because the protocols are so intricate, they are also extremely expensive (Nair, 2014). This is a big limitation on forensics because it is hard to obtain new information about ancient human life in aDNA (ancient-DNA). Most of the greatest claims about DNA survival have been because of cross-contamination in the lab, which means the actual aDNA was wasted and not actually what was observed (Willerslev and Cooper, 2005). The study in this experiment is useful in the big picture because it shows that even with high attempts at precision, the results were still not accurate in PCR and DNA extraction, so it would likely be a good idea to rule out this procedure while using aDNA.