Question

1. Why can’t the DNA from a crime scene sample be extracted, fragmented and immediately run on in an Argos rose gel electrophoresis?

2. Why is it necessary to stain the electrophoresis gel before measuring it?

Answer 1

1) Samples are collected from different sources during crime investigation. The samples may be contaminated with bacteria or other species DNA. DNA sample may not be sufficient for PCR analysis. The cells with DNA may be present on another object, such as saliva and body fluids present on the victim. Hence, the sample has to be first processed before it can be sent for DNA analysis. The sample has to be stored in a cool container before DNA analysis. DNA is then sent for analysis. The DNA is first extracted or isolated from the sample. Depending on sample amount, various methods can be applied. Samples from embalmed bodies, pathology, or fetal tissue stored in formaldehyde are generally not suitable for DNA analysis. There are four steps in DNA analysis:

1) Common inhibitors are hemoglobin and indigo dyes from denim in the samples. These have to be removed from the sample. The sample has to be lysed in buffer solution and denatured proteins/lipids removed by centrifugation. DNA is then separated by passing through a column. The DNA is purified to remove other contaminating substances. Finally, the DNA is quantified to calculate the amount of DNA present.

2) The sample may contain DNA from other sources such as bacteria. A step called quantitation is performed where the quality and quantity of human DNA is assessed. A sequence detection system is used for this analysis.

3) The DNA has to be amplified as the amount of DNA may not be sufficient for analysis. PCR is carried out to amplify certain sequences of DNA and then the PCR product can be visualized by capillary gel electrophoresis. Short tandem repeat sequences are sequences that have 2-13 nucleotide that are repeated several hundred times in a row. Such repeats vary among individuals and can be used for DNA analysis. The STR are amplified using PCR and then identified by agarose electrophoresis.

As the DNA may be contaminated with other sources, is limited in amount, it cannot be directly run on the gel after extraction.

2) The DNA or protein are separated by electrophoresis on basis of molecular weight. Hence, smaller molecules will run faster than the larger molecules. The bromophenol blue dye will only indicate how far the smallest molecule has run. It will not identify the position of the other fragments. Therefore, in order to assess the distance travelled by other fragments and to identify/visualize the other fragments, the gel has to be stained. DNA is visualized by ethidium bromide staining. Ethidium bromide intercalates between the DNA when the fragments are run on the gel. Exposure to uv rays will cause ethidium bromide to fluoresce and exhibit a red orange color, which can be easily seen. For protein blue, dyes such as Coomassie blue bind to protein and can be seen as blue color across a clear background after destaining.