Development of a PCR-based method for detection of nod a gene and diversity studies of selected photosynthetic Rhizobia.

Date

3-2005

Degree

Bachelor of Science in Biology

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Gem D. Encarnacion

Co-adviser

Neilyn Ona-Villa

Abstract

A non-nodulating bacterium Escherichia coli, one non-photosynthetic and six photosynthetic rhizobia were used for the detection of nod a gene and for the diversity studies using Internal Transcribed Spacer (ITS) region and Repetitive Extragenic Palindomic (REP) sequences. Different extraction techniques were used to extract the genomic DNA of the bacterial isolates; these were Cetyltrimethyl Ammonium Bromide (CTAB) method. CTAB treated with RNAse, Boiling method and Freeze-thaw method. Boiling and freeze thaw method produced genomic DNA approximately less than 25 ng/ul as composed to 50 ng/ul of DNA concentration from CTAB method. Leastsmeared DNA comes from CTAB treated with RNAse. Total genomic DNA isolated from all photosynthetic rhizobia were used as template for PCR amplification of the nod A gene. The expected size of 666 bp was obtained from all the photosynthetic strains and a slightly longer band (approximately 750 bp) from the non-photosynthetic strain pLBI. No amplicons were obtained from the non-nodule forming bacterium, E coli. DNA directly extracted from the stem nodule when used as template did not produce any amplicons. With the use of DNA from the root of nodules, an amplicon longer than the expected product was obtained. When the genomic DNA extracted using different techniques was used as template for PCR amplication, different bending patterns for ITS were observed. Based on the banding patterns produced, no two isolates were exactly the same. The dendogram created using the banding pattern data of the 25 cycles ITC-PCR showed groupings similar to that obtained by Ladha and So (1994) which made use of phenotypic characteristic and Cantera et al. (2002) which made use of sequence analysis of nod A and nif genes. No PCR amplicons were produced using the REP-PCR

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

Thesis

Document Type

Thesis

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