Applying the Method of DNA Barcoding to Identify Ilex x attenuata, Truong, Julia
College of Science and Mathematics
Professor: Dr. Brenda Whaley
DNA barcoding is a technique that uses a standard region of an organism’s DNA to sequence and identify the corresponding genus or species. An ideal genomic region for DNA barcoding should be able to be amplified by polymerase chain reaction using common primers, demonstrate high quality bidirectional sequencing with minimal editing, and provide maximal discriminating power among species. The rbcL gene, which is found on the chloroplast DNA and codes for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCoO) long chain, offers these important qualities, and is therefore used as the DNA barcode for plants.
To begin, a single leaf was obtained from the plant of interest, which is located at 29.861011°N and -95.600770°W, and pictures were taken of both the individual leaf and the whole plant. A small section of the leaf was used to isolate the DNA from the plant tissue. The rbcL gene was then amplified by polymerase chain reaction (PCR) using two different primers. Thermal cycling made copies of the rbcL gene by denaturing, annealing, and extending the DNA region. Next, gel electrophoresis was used to analyze the amplification products for correct length and purity. Gel staining allowed the PCR samples to be visible, which were photographed and sent along with a sample of the PCR product to a sequencing company. After receiving the sequences of the gene, they were used to perform a BLAST search. By using the closest match for the rbcL sequence identified by BLAST and evaluating the leaf anatomy, the unknown plant was identified as Ilex x attenuata, also known as Savannah holly.
