Methods for Isolation and Sequencing of Topoisomerase II gene in Gossypium hirsutum

Abstract

Topoisomerase II (topo II) is an enzyme that is essential in cell division. Topo II alters DNA supercoiling, thus affecting every aspect of DNA function. Gossypol is a chemical produced by Gossypium hirsutum that is known to inhibit topo II. G. hirsutum has two complete genomes (an A and a D), making it difficult to sequence any particular gene.

Through data bank queries and BLAST nucleotide searches, probable partial cDNA sequences of topoisomerase II for G. hirsutum for both A and D genome were identified. A common start primer was used for both genomes. The reverse primers were specific for the 3’ end of the coding sequence for topo II. A middle primer was design based on topoisomerase II amino acid sequences near the middle of the protein from several green plant.

Gossypim hirsutum seeds were germinated and allowed to sprout. Total RNA was taken from the leaves. A cDNA library was made from the total RNA. Polymerase chain reaction (PCR) was used to amplify the topoisomerase II sequence. Bands at sizes of around 4000 (start primer - reverse primer) and 2000 (middle primer – reverse primer) base pairs were expected. The PCR reactions resulted in products of these sizes, and the PCR products were recovered from the gel. Extracted DNA was put into a cloning vector and then the DNA was sequenced. Cloning and sequencing produced no obvious topo II sequences, possibly because the extracted DNA had very low concentrations.