Question

I. Describe one DNA extraction method. In your description compare and contrast the method you chose with the other DNA extraction methods. Additionally, describe what types of forensic samples are better suited for this type of extraction and why. a)Given the three types of DNA techniques (STR, mtDNA, and Y-STRs) briefly compare and contrast all of them and in your opinion state why you believe one to be better than the other. YOU MUST SUPPORT YOUR ANSWER WITH AN EXAMPLE (REAL OR MADE-UP).

Answer 1

DNA can be extracted in many ways : organic ( variations of phenol-chloroform ), inorganic chelex or silica method, solid phase extraction methods, differential extractions.

In organic extraction, the cells must be lysed using gentle heat to disrupt the membranes with the assistance of SDS(sodium dodecyll sulfate) and DTT(dithiothreitol). Proteinase K must be added to digest the proteins, including nucleases. In the second step of the extraction, the DNA must be separated from the other cellular components using the phenol-chloroform-isoamyl alcohol (PCIA) reagent. When the PCIA is added 2 phases appear : the organic ( bottom) and the aqueous ( DNA-containing) phase (top) after centrifugation. The DNA containing portion is removed by pipetting of the top layer. DNA is more soluble in the aqueous state comparing to organic phases. It is important to remove all of the organic extraction agents because phenol will degrade DNA.Phenol has a density of approx. 10 g/mL and can easily invert with the aqueous phase. Lastly in the third step, the DNA can be filtered and concentrated by centrifugation through a Microcon, Centricon or Vivacon 100 filter in which the DNA is captured on the membrane while all other aqueous components including protein fragmants, residual organics and potential PCR inhibitors are allowed to pass through the membrane. The DNA is eluted by inverting the filter and centrifuging using a desired volume of elution buffer or nuclease free water. Using the filter the DNA is concentrated in a small volume for a subsequent amplification steps.

This method yielda double stranded DNA extract and the highest yield but requires multiple tube changes which increase the possibility of contamination error.

Bone, teeth, hair roots , blood and bone marrow aspirates will be the good samples as the technique helps to get PCR inhibitors free high yeld of double stranded DNA from a single source sample.

Inorganic chelex method is a simple and cheap one tube extraction process in which Mg2+ binds to resin beads and yields a sungle stranded DNA product. Solid phase extraction method is simple extraction process in which DNA binds to paramagnetic or silica beads. Differential extraction is a multistep process used to separate sperm from other cells using DTT; used for analyzing biological evidents from a sexual assault cases.

STR or short tandem repeat is used to compare specific loci on DNA from two or more samples. It is a microsatellite which measures exact number of repeating units. As unrelated people have different number of repeating units STRs can be used to discriminate between unrelated individuals.

A Y-STR is a short tandem repeat on the Y chromosome; used in forensic, paternity and genealogical DNA testing. Y-STRs taken from male chromosome and provide a weaker analysis than autosomal STRs because the Y chromosome is practically identical in only paternal line. mtDNA analysis is used to create DNA profiles from types of evidence that might not be appropriate for other DNA analysis techniques. The technique utilizes DNA found in the mitochondrion.

I think mtDNA technique is the best as sometimes sample can be old ( very old teeth, bone or hair shaft) and will no longer have nuclear material in the cell which poses a problem for other types of DNA analysis. However mitochondrial DNA can be removed, thus having ramifications for cases that were not solved for many years. The mtDNA will be the same from women to daughter as it is passed from egg cells and thus can be useful to investigate for a missing person. Moreover, mtDNA can be useful for analyzing evidence degraded through issues such as improper storage.