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Forensic Analysis of Canine Samples

        By comparing the canine DNA found on Professor Millstone and at the crime scene to that of the canines that belong to two suspects, Dr. Hedd and a disgruntled student, reasonable and justifiable claims about the individual responsible for Professor Millstone's death can be made. Polymerase chain reaction (PCR) as well as polyacrylamide (PAGE) and agarose gel electrophoresis are critical DNA analytical techniques that can be used to amplify certain regions of DNA using loci-specific primers and characterize the amplified DNA by separating fragments according to number of base pairs (size, weight), respectively. STR-PCR specifically amplifies loci containing two-to-ten nucleotide sequence repeats, also known as short tandem repeats (STR). When STR-PCR products are analyzed via gel electrophoresis, the variability in the number of repeats creates unique DNA profiles which can then be used as a means of identifying or distinguishing individuals.

DNA was extracted from the bulbous ends (nuclei degrade as hair cells are keratinized) of canine hairs found on the victim, a saliva sample from the bite on her hand, hair and saliva samples from Dr. Hedd's female dog, and hair and saliva samples from the student's parent's male puppy. Also, isolated DNA from the hairs of a known female and male dog were used as a control to provide a standard of difference that could be used to better assess the similarity between the samples collected from the suspects and those collected at the crime scene. For the purpose of this lab, three canine-specific hypervariable nuclear loci (PEZ 2, PEZ 15, and VWF.X) were amplified via PCR and characterized with high resolution PAGE. Two sex-specific loci, SRY and CHR.X, were also amplified and visualized on an agarose gel. In addition to nuclear DNA, a ~500bp control region (WD3/WD6), not present in humans, containing varying numbers of 10-bp tandem repeats was also amplified and characterized via PAGE for each canine sample collected. 

         Uniqueness between these samples only occurred within DNA samples of sex specific loci ???????? , indicating our DNA profiles differed only in the sex specific loci of the samples found (Figure 4).  mtDNA control region fragments revealed that samples collected from Millstone, the student, and Hedd were all related and therefore from the same maternal line.  The range of mtDNA fragment sizes from evidence samples ranged from 437-463 bp in size but relative to the control mtDNA fragments from known male and female dogs,we can  can infer that the evidence mtDNA samples are the same size and therefore from the same maternal line (Table 1).  The control mtDNA fragments were much ?? larger than the mtDNA samples from Millstone, the student and Hedd indicating these evidence samples are within the margin of error (Figure 3).  Our findings  findings support a constant profile independent of tissue type because all cells contain mitochondria and therefore mtDNA.  All samples collected regardless of the source contain an approximately 500bp control region. WHAT ABOUT THE PATTERN OF BANDING AT THE APPROPRIATE BP? DO YOU KNOW ANYTHING ABOUT THIS FROM THE HANDOUT? REFERENCE HERE? Nuclear   Nuclear DNA PEZ2, PEZ15 and VWF.X were vague  ??? and  vague and samples from the student’s dog saliva and Hedd’s dog hair was lost.    WHAT SHOULD YOU SEE HERE? However   What should be seen here is a distinction between nuclear DNA, specifically nuclear DNA from either Hedd's or the students's dog that matched the samples found on Millstone.  However comparing duplicate gels of the same samples, we concluded that all three DNA sources fit the same DNA profile and therefore shared the same DNA fragment sizes (Figures 1, 2). FIGURES SHOULD BE ADDRESSED IN NUMERICAL ORDER...1,2,3,4. NICE TABLE. NEED TO ADD FORMATTING TO USE FOOTNOTE ON BOTTOM OF TABLE. 

        Theoretically, knowing the student owns a male dog and professor Hedd owns a female dog, we must use the sex specific gel containing the sex specific loci DNA samples because mtDNA and nuclear DNA samples all fit the same genetic profile.  The samples from Millstone’s body are male ARE THEY??? I DONT AGREE...THE DATA IS MIXED AND INCONCLUSIVE! possibly (though inconclusively) male and knowing the student owns a female dog, we can assume the student was at the scene of Millstone’s death. CONTROLS HERE? However The controls in Figure 4 are clear and indicate both the bands necessary to identify a male and female dog.  However, the results are faulty HOW? and due to missing nuclear DNA evidence and further testing needs to be done in order for the evidence to completely support a verdict in court.  Further testing includes certain thoroughness and carefulness when handling gels, as they are fragile,BUT THE GELS WORKED FINE...WHAT ELSE NEEDS TO BE DONE?? WHAT WAS THE ISSUE?? WHAT ELSE NEEDS TO BE DONE??  and being weary of contaminating evidence HOW?.  Sep through sterilization.  These factors can be sources of error in this experiment as well as hair samples being cut in the bulb region rather than the shaft region, which may have occurred.  Also, the stains used could have affected the vividness of the samples when ran through the gel as well as the percent of acrylamide and agarose used, dulling the bands from the DNA samples.  This means that the samples could have been present but the parameters of the experiment affected their visualization.  The lack of supporting evidence from laboratory error decreases the overall conviction using the gels as support.  Other controls that could have aided in the experiment include mtDNA and nuclear DNA from the same species of dog the samples were obtained from.  Separating samples throughout more gels would allow for an easier analysis of data as well.  The likelihood that the mtDNA of a dog matches another dog by chance is 8%. SIG FIGS?? WHAT ABOUT THE NUCLEAR STRS SPECIFICALLY ASKED IN QUESTION?? Therefore7.89%.  The likelihood the nuclear DNA of a dog matches another dog by chance was given and is .193%.  Therefore, the probability of having two random dogs with matching nuclear loci (PEZ 2, PEZ 15, VWF.X), mitochondrial control regions (W 3/6), and the same sex is approximately 0.008% or 8 out of100 1,000 dogs. (??? TRY AGAIN BUT DEFINITELY USE THE 1 IN HOW MANY DOGS HERE!)

ERROR ANALYSIS?

ARE THERE ANY CONTROLS THAT COULD BE INCLUDED TO HELP IN THIS INVESTIGATION?

% ACRYLAMIDE AND AGAROSE IN GELS? STAINS USED?

WHAT DO YOU SEE ON THE GELS WITHOUT DRAWING CONCLUSIONS IN THE LEGENDS?

REFERENCES??

 

http://en.wikipedia.org/wiki/Mitochondrial_DNA#Origin

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