The National Association of Biology Teachers recognizes the importance of laboratory activities using human body samples and has developed minimum safety guidelines to minimize the risk of transmitting serious disease. ("The Use of Human Body Fluids and Tissue Products in Biology," News & Views, June 1996.) These are summarized below:

1. Only collect samples from students under your direct supervision.
2. Do not use samples brought from home or obtained from an unknown source.
3. Do not collect samples from students who are obviously ill or are known to have a serious communicable disease.
4. Have students wear proper safety apparel: latex or plastic gloves, safety glasses or goggles, and lab coat or apron.
5. Supernatants and samples may be disposed of in public sewers (down lab drains).
6. Have students wash their hands at the end of the lab period.
7. Do not store samples in a refrigerator or freezer used for food.

The mouthwash method of DNA isolation does generate liquid waste; however, the risk of spreading an infectious agent is much less likely than from natural atomizing processes, such as coughing or sneezing. Several elements further minimize any risk of spreading an infectious agent that might be present in mouthwash samples:

1. Each experimenter works only with his or her sample.
2. The sample is sterilized during a 10-minute boiling step.
3. There is no culturing of the samples that might allow growth of pathogens.
4. Samples and plasticware are discarded after the experiment.

Student particpation in this experiment raises real-life questions about the use of personal genetic data:

1. What is my DNA sample being used for?
2. Does my DNA type tell me anything about my life or health?
3. Can my data be linked personally to me?

There is concensus that a human DNA sample should be obtained only with the willing consent of a donor, who understands the purpose for which it is being collected. Thus, the experiments should be explained ahead of time and students given the option to refrain from participating. (Some teachers may wish to have parents sign a consent from, such as those filled out for a field trip.) There is also consensus that a DNA sample be used only for the express purpose for which it is collected. Thus, student DNA samples should be thrown away after completing the experiments in this unit. (In fact, the cheek cell samples are not stable enough for long term storage.)

The Alu PV92 insertion and mt control region polymorphisms used in these experiments were specifically selected because they are phenotypically neutral. Neither locus encodes a protein, nor has any known relationship to disease states, sex determination, or any other phenotype.

Even though there is no chance of disclosing phenotypic information about the experimenters, all student polymorphism data stored at our Allele Server and Sequence Server sites are anonymous. Online submission forms identify students only by number and have no entry fields for personal identifiers. We recommend that each student select a four-digit personal identification number (PIN) and label their experiment with this number. Under this system, there is no chance that a student can ever be linked to his/her sample in the database. Alternately, students can be assigned a sequential number, with no permanent key maintained by the teacher.

All polymorphisms are inherited in a Mendelian fashion and can give indications about family relationships. The PV92 polymorphism has an inherently low information content – usually there are at least several parental genotypes that could account for an observed student genotype. On the other hand, mitochondrial (mt) DNA genotypes are explicit records of maternal inheritance that are usually unique to each family. Since mt DNA is inherited exclusively from the mother, there is no chance of showing nonpaternity, which is the most frequent problem uncovered by DNA testing. However, inconsistent mt DNA types between siblings and/or mother could suggest adoptive situations.

To avoid the possibility of discovering inconsistent mt DNA inheritance, we recommend that you do not generate genotypes from family members. However, in a formal sense, a single data set – even for mt sequence – cannot definitively prove or disprove relatedness for several reasons:

• Student samples can be easily mixed up, by mislabeling or misloading, at any point in the multi-step procedure – DNA isolation, PCR reaction, and gel electrophoresis. A forensic laboratory would use approved methods for maintaining "chain of custody" and for tracking samples, which insure that a specific result is from a specific sample. However, we do not recommend instituting rigorous controls to insure sample identity. In an educational setting, "limited sloppiness" provides a safety valve for inconsistent results.

• A single "read" of mt DNA sequence is not definitive. Sequence errors do occur during amplification, sequencing, and "base calling" by the sequencing software. Finished DNA sequences submitted to DNA databanks have typically been resequenced 2-8 times to clarify any discrepancies in single reads. At a minimum, a reverse read of the complmentary DNA strand would be required for any definitive determination.

• New mutations and heteroplasmy occasionally occur in mt DNA lineages. A new mutation may occur in one family member, resulting in a different nucleotide at that position. A new mutation may also lead to a situation know as heteroplasmy – when cells harbor two populations of mitochondria, each with a different nucleotide at a particular position.