Document Type

Honors Project

First Advisor

Dr. Stephen Baron

Degree Award Date

Fall 2007

Keywords

Polymerase Chain Reaction, Cloning, sequencing, Polyhydroxybutyrate Depolymerase Gene, Streptomyces sp. 5A, DNA probe, southern blotting technique, Diverging non-degenerate primers, inverse PCR

Disciplines

Biology | Cell and Developmental Biology | Molecular Genetics

Abstract

A .500 base pair (bp) segment of the polyhydroxybutyrate PHB depolymerase gene (phaZ) of Streptomyces sp. 5A was amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Homology searches suggested that this segment (A20TG) comprised about 40% of the length of phaZ. Specific primers for A20TG were then used in PCR to construct a phaZ-specific DNA probe. The probe was used in the southern blotting technique in order to identify restriction fragments suitable for cloning the remainder of phaZ and its upstream region; however, results from this method could not be analyzed and interpreted with certainty. Diverging non-degenerate primers complementary to A20TG were used in an alternative method, inverse PCR, in attempts to clone the remainder of the gene. We have obtained PCR products which will be cloned and sequenced for this purpose.

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