Beyond Labz

The overall goal of the Molecular Biology laboratory is to give students the opportunity to produce DNA sequences of amplified segments and genes and then compare those sequences among related species. To produce these DNA sequences within the virtual laboratory, students must have the opportunity to prepare DNA samples from selected species, run a PCR amplification experiment, confirm the amplification using gel electrophoresis, and finally determine the sequences of the amplified products. By necessity, certain shortcuts and simplifications have been made in order to produce a workable, broad-based simulation. Given below is a brief outline of these simplifications.

DNA. We have created a model genome for each species available in the molecular laboratory, which is comprised of a set of actual gene sequences acquired from GenBank interspersed with random sequences of DNA. Obviously because of size and speed constraints we could not provide genomes even remotely close to full size, however we have attempted to provide a self-consistent set of genes that can be compared among our collection of species whenever possible. Most species have 10 to 15 genes within their model genome, but some are as few as 4 and many species have more than 20 genes. Most of the gene selection was determined by what was comprehensively available on GenBank.

Preparing Samples. Samples are prepared for PCR experiments by adding DNA from a species, Taq, either tagged or untagged nucleotides, and the correct primers required to amplify the target gene. In a real experiment the correct amount of each reagent is important, but in this simulation the focus is on having the students know what they need to add and not how much. It is assumed that the correct amount is added each time. In the case of a DNA sample, it is assumed a very small number of strands are added to the tube where the number can range from 7 to 15 strands. Any required sample preparation to extract the DNA is assumed when the DNA is added.

Primers. The primer tab in Live Data contains a list of all primers that are needed to amplify any gene that is included in the model genome of all DNA samples that have been added to the tubes. In addition to all of the actual primers a set of random primers are also included to make primer selection more realistic. The Primer Window also allows custom primers to be created. The list of recommended primers was generated by taking the 3’ and 5’ sequences at the beginning and end of each gene in the model genome.

PCR Simplifications. In the PCR experiment we have made several simplifications: (1) A successful primer annealing requires an exact match of the primers in the tube and the matching sequence on the DNA strand. No base mismatches are allowed. (2) Actual primer annealing temperatures vary by the GC content, however in the simulation we assume the annealing temperature is independent of GC content and we assume a rather broad range of temperatures for a successful annealing. (3) The simulation defines a range of temperatures and dwell times for each step in the PCR process. If the user selects a temperature or dwell time outside this window then the entire PCR process is not successful.

Given these simplifications, the rest of the PCR simulation is realistic. For example, if the selected primers do not match then amplification will not occur. If the primer is too short or not unique then multiple strands will be amplified. If the separation between the 3’ and 5’ primers is too long then amplification will not occur. If the correct number of cycles is not used then insufficient amplification will occur.

Gel Electrophoresis. In the gel electrophoresis experiment, students pipet amplified product into one of 10 different wells in the gel and then apply a voltage across the bed to separate any DNA mixtures into bands according to their mass or number of base pairs. The gel matrix is a crossed polymer and the gel performance is modeled after actual gels. A gel experiment requires a tracking dye to monitor the position of the bands as they move down the gel and a fluorescent tag that attaches to the DNA to mark the position of the products. In the simulation, bromophenol blue is used as the tracking dye and SYBR is used the marking dye. Both are added automatically when samples are placed in the wells. A standard set of markers are also included in wells #1 and #10 in order to calibrate any bands isolated in the other 8 wells.

Automatic Sequencer. Actual DNA sequencers are sophisticated pieces of equipment and require a unique set of reagents. The virtual automatic sequencer used in the molecular laboratory is completely automated, provides sequence information at the rate of one base pair every 30 seconds, and requires only that the student has amplified enough product to provide a sufficient quantity for the sequencer reactions. No other sequencer reagents are required.

Species and Gene List. The following is a general list of genes that we have attempted to include in the model genomes of each species. This list comprises the genes we attempted to identify and include, but the actual genes that make up each model genome were governed by what was found on GenBank. Following this list, a specific list of genes and accession numbers for the actual genes included for each species is also included. Information on each gene, including the actual sequence used to create the model DNA, can be found by visiting The National Center for Biotechnology Information and searching for the selected gene using the accession number provided below.

General List of Genes

• 5S is a ribosomal RNA. It is a component of the large subunit of the ribosome in all organisms.
• 12S is a mitochondrial ribosomal RNA present in all animals. Mutation in this gene has been associated with hearing loss.
• 16S is a ribosomal RNA. It is a component of the small subunit of the ribosomal in all prokaryotes.
• 18S is a ribosomal RNA. It is a component of the small subunit of the ribosome in all eukaryotes.
• 28S is a ribosomal RNA. It is a component of the large subunit of the ribosome in all eukaryotes.
• Actin is a highly conserved globular protein present in eukaryotes. It is involved in the formation of filaments that are a major component of the cytoskeleton. Interaction with myosin provides the basis of muscular contraction and many aspects of cell motility.
• aspC is the gene encoding aspartyl-tRNA synthetase protein. It catalyzes a two-step reaction, first charging an aspartate molecule by linking its carboxyl group to the alpha-phosphate of ATP, followed by transfer of the aminoacyl-adenylate to its tRNA
• atp6 is the gene that encodes the F0 subunit 6 of ATP synthase. ATP synthase is a transmembrane protein that plays the key role in translocating protons across the membrane.
• atpB is a gene that encodes for the beta subunit of ATP synthase. ATP synthase is a transmembrane protein that plays the key role in translocating protons across the membrane.
• clpX is the gene that encodes for the ATP-binding subunit of the ATP-dependent Clp protease. Protease is an enzyme that breaks down proteins by hydrolyzing the peptide bonds between amino acids.
• CO2 gene encodes for subunit 2 of cytochrome oxidase. It is a transmembrane protein and functions as the last enzyme in the electron transport chain.
• CO3 gene encodes for subunit 3 of cytochrome oxidase. It is a transmembrane protein and functions as the last enzyme in the electron transport chain.
• COI gene encodes for subunit 1 of cytochrome oxidase. It is a transmembrane protein and functions as the last enzyme in the electron transport chain.
• cytB gene encodes for the protein called cytochrome B. It is the main subunit of respiratory enzyme complex such cytochrome bc1 and b6f.
• EF1 is a transcription factor. It binds to specific DNA sequences and controls transcription of DNA to mRNA.
• glnA gene encodes for glutamine synthetase enzyme. It plays an essential role in nitrogen metabolism by catalyzing the condensation of glutamate and ammonia to form glutamine.
• gltA gene encodes for citrate synthase enzyme. It is localized within mitochondrial matrix of eukaryotic cells and plays an important role in Kreb’s cycle.
• glyA is the gene that encodes for the enzyme called glycine/serine hydroxymethyltransferase. It belongs to the transferases family of enzymes.
• Hist3 encodes for histone cluster 3. Histone proteins play an important role in gene regulation by forming the spool around which the DNA winds.
• matK encodes for maturase K protein. It assists in splicing the introns in a gene.
• ND1 encodes for NADH dehydrogenase subunit 1. This enzyme catalyzes the transfer of electrons from NADH to coenzyme Q in the electron transport chain.
• P450 encodes for cytochrome P450 gene. These are proteins that contain the heme group and carry out the electron transport chain.
• PAL gene encodes for peptidylamidoglycolate lyase protein. It’s an enzyme that catalyzes the reaction of peptidylamidoglycolate into peptidyl amide and glyoxylate.
• Pgm is the gene that encodes for phosphogluconate mutase enzyme. It catalyzes the bidirectional interconversion glucose-1-phosphate and glucose-6-phosphate via a glucose 1,6-diphosphate intermediate, an important metabolic step in prokaryotes and eukaryotes.
• RNII encodes for RNA polymerase II. It catalyzes the transcription of DNA to mRNA.
• TPM is the gene that encodes for proteins in trapezius muscle lean area. The trapezius muscle extends longitudinally from the occipital bone to the lower thoracic vertebrae and laterally to the spine of the scapula.
• TPN is the gene that encodes from transposase enzyme. It binds to the ends of a transposon and catalyzes its movement to another part of the genome.
• Trnl gene encodes for transfer RNA. tRNA transfers amino acids to a growing polypeptide chain at the ribosomal site of protein synthesis.

List of Species and Genes