Exercises

These free exercises use the open-source modules of the Cybertory platform. Please contact us if you would like to install the programs and data sets on your own server. Using your own server may be more reliable for classroom use.


Paternity Testing

In this exercise, we use simulated experiments to test the DNA of six individuals: a mother, her three children, and two different men who may be the fathers of the children. DNA samples are used as templates in Polymerase Chain Reaction (PCR) experiments to determine which variants each child has at certain genetic positions. A child inherits one variant from each parent, and we can usually match one up to a variant in the mother. This means the other one had to come from the father. If neither one of a possible father's genetic variants match, then he is not the father. If we examine enough genetic positions, it is very likely that we will be able to rule out any man who is not actually the father of a particular child.

When you have finished the experiment, you can look at a family tree of the individuals to confirm their relationships, and check your answers.

Technologies: This exercise uses the Cybertory PCR simulator with genomes of simulated individuals. Results are preseted as gel images.

Time required: approximately 2 hours.


Forensic Identification

We will do simulated experiments to test the DNA of several individuals. These DNA samples simulate materials recovered from crime scenes (blood, semen, skin, etc.); your job is to try to identify the individual matching the sample, based on PCR analysis of STR loci.

When you have determined the number of repeats for each allele in your sample, you can search a database to see if your sample matches any known criminal, or other samples from unsolved crimes.

Technologies: This exercise uses the Cybertory PCR simulator with individual human genomic templates, and named STR primers.

Time required: approximately 2 hours.


Computerized STR Analysis

STR analysis is primarily a matter of testing for the presence or absence of products of particular sizes, representing the various alleles. This exercise uses analysis software to help you organize and manage data from PCR results.

Technologies: This exercise uses the CybertoryTM PCR simulator with simulated individual human genomes.

Time required: approximately 2 hours.


PCR Primer Design

Given the sequence of a bacterial gene, you will learn to design a pair of PCR primers to amplify a particular target region. You will test the effects of reaction conditions on reaction yield and specificity for various primers. Finally, you will test your primers on different bacterial strains, for which you do not have the genomic sequence, to see if they will amplify related genes.

Technologies: This exercise uses the Cybertory PCR simulator with the E. coli genome.

Time required: approximately 2 to 4 hours.


Shotgun Sequencing

Dideoxy chain-termination sequencing depends on synthetic DNA primer sequences to initiate the reaction. These primers must match a portion of the template whose sequence we are trying to determine. This gives us a 'chicken and egg' problem of needing to know a bit of the template sequence before we can read more of it.

One way to start sequencing an unknown sequence is to make a recombinant clone putting the unknown insert in a vector of known sequence. Then primers from the vector can be used to begin reading the sequence of the insert. Once a portion of the new insert sequence is known, we can use that to design a new primer to let us read further. This process can be repeated until the whole insert is sequenced. This 'primer walking' process is inherently sequential, since each step must be completed before the results can be used to design the primer for the next step.

Shotgun sequencing is an approach that lets us run large numbers of reactions in parallel, rather than in series. Rather than using primer walking through one large insert, we randomly fragment the insert to create a library of smaller fragments. A large number of these clones are chosen at random, and sequenced in parallel using primers matching the vector. The sequencing results are then 'assembled' on the computer into a contiguous sequence of overlapping fragments. This approach essentially trades much of the laborious laboratory work for a puzzle to be solved on the computer, and turns out to be much faster than pure primer walking.

Technologies: This exercise uses the Staden sequence data management software for assembly of reads into contigs. Using the sequence simulation module, students must design custom sequencing primers for primer walking through regions not adequately covered by random clones, and to resolve ambiguities.

Time required: approximately 4 hours.


HIV Genotyping

Untreated infection with the Human Immunodeficiency Virus leads to AIDS, a deadly and widespread disease. Though many effective drugs are available, the virus mutates rapidly, and drug resistance evolves quickly. In this exercise, you will use simulated DNA sequencing to determine the differences from the standard using Staden's trace-subtraction function. This list of mutations will let you query the online Staford HIV Database to predict the drug resistance and susceptability of this viral strain. Building on the earlier shotgun sequencing exercise, we perform 'resequencing' by aligning reads from clinical samples against a standard sequence. We also take advantage of the Staden sequence data management software to perform trace subtraction to detect variations from the standard.

Time required: approximately 3 hours.