US 5,849,491 A · Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby

Abstract

Xylanase DNA is recovered from soil by PCR amplification using degenerate primers. Because of the complexity of the soil samples, it is likely that the recovered product will include more than one species of polynucleotide. These recovered copies may be cloned into a host organism to produce additional copies of each individual species prior to characterization by sequencing. Recovered DNA which is found to vary from known xylanases can be used in several ways to facilitate production of novel xylanases for industrial application. First, the recovered DNA, or probes corresponding to portions thereof, can be used as a probe to screen DNA libraries and recover intact xylanase genes including the unique regions of the recovered DNA. Second, the recovered DNA or polynucleotides corresponding to portions thereof, can be inserted into a known xylanase gene to produce a recombinant xylanase gene with the sequence variations of the recovered DNA.

Claims7

Dependent claims

A method for recovering xylanase-encoding DNA from soil, comprising the steps of:

(a) treating a soil sample to render DNA in the soil accessible for hybridization with oligonucleotide primers,
(b) combining the treated soil sample with first and second amplification primers in an amplification reaction mixture, said first and second amplification primers hybridizing with conserved regions of the sense and antisense strands respectively of a gene encoding a xylanase and flanking a region of interest in the gene,
(c) thermally cycling the amplification reaction mixture through a plurality of cycles each including at least a denaturation phase and a primer extension phase to produce multiple copies of the region of interest flanked by the first and second amplification primers, and
(d) recovering the copies of the region of interest from the amplification reaction mixture, wherein the method further comprises the step of screening a treated soil sample to isolate full length DNA encoding a complete xylanase using a probe having a sequence which is the same as or fully complementary to at least a portion of a recovered copy of the region of interest, said portion being different from the reference xylanase sequence given by SEQ ID NO 23.

1 more element

Dependencies are inferred from the claim text.

Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby

Life of this patent

Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby

Life of this patent

Abstract

Claims7