DNA barcoding for Ligustrum japonicum, Bhatt, Asha
College of Science and Mathematics
DNA barcoding is a molecular tool that uses a marker within an organism’s DNA sequence to identify it with a particular species group. The main purpose of DNA barcoding is to identify an unknown sample with a known specie group. The distinct differences between the genomic regions within the plants are adequate to provide specific specie information. However these genomic regions are still conserved to the point that generic primers can be used to amplify DNA using the polymerase chain reaction method. The specific DNA barcodes for our chosen plant contains the sequence for the gene that codes for ribulose-1,5-bisphoshpate carboxylase/oxygenase (RuBisCO), which is found on the chloroplast DNA. This gene is called rbcL. DNA barcoding was performed on a leaf tissue obtained from a plant garden at the following address, 4107 Vaughn Creek Court, Sugar Land, Texas. A small amount of the plant tissue was obtained and placed in nuclei lysis solution to dissolve the membrane bound organelles. The samples were then incubated and placed in RNase solution, protein precipitation solution, and isopropanol. PCR was then performed to amplify about 600 bases from the 5’ end of the rbcL gene. The primers used for rbcL were:
5’-TGTAAAACGGCCAGTATGTCACCACAAACAGAGACTAAAGC-3’
(forward primer – rbcLaf-M13)
5’-CAGGAAACAGCTATGACGTAAAATCAAGTCCACCTCG-3’
(reverse primer – rbcLa-revM13)
After PCR, gel electrophoresis was done to analyze the PCR products. Gel electrophoresis analyzes the PCR products for correct length and purity. The gels were then stained and photographed. The stained pictures and 10 µL of the PCR product was sent to GeneWiz Inc2 to be further analyzed and sequenced. A BLAST search was then used to determine the specie with the closest match to the obtained rbcL sequence. Based on the BLAST results the plant of interest was identified as Ligustrum japonicum.
