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

• US 5,849,491 A
• Filed: 09/20/1996
• Issued: 12/15/1998
• Est. Priority Date: 09/22/1995
• Status: Expired due to Fees

• Current Assignee: Cubist Pharmaceuticals, Inc. (Merck & Co., Inc.)
• Original Assignee: Terragen Diversity, Inc.
• Inventors: Yap, Wai Ho, Warren, R. Antony J., Radomski, Christopher C. A., Seow, Kah Tong
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/716,942
• Time in Patent Office: 816 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 435/5, 536/23.1, 536/24.3-24.33
• US Class Current: 435/6.13
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01032 Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase

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.

Claims

1. 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.

2. The method according to claim 1, wherein the probe has a sequence which is the same as or fully complementary to at least a portion of any one of Seq. ID Nos. 3 through 22 that is different from the reference xylanase sequence given by Seq. ID. No. 23.

3. The method according to claim 1, wherein the probe has a sequence which is the same as or fully complementary to any one of Seq. ID Nos. 3 through 22.

4. A method for recovering a xylanase gene from soil, comprising the steps of:

(a) combining a treated soil sample in which soil DNA is rendered accessible for hybridization with a polynucleotide probe having a sequence which is the same as or fully complementary to at least a portion of any one of Seq. ID Nos. 3 through 22 that is different from the reference xylanase sequence given by Seq. ID. No. 23; and

(b) isolating DNA that hybridizes with the probe from the treated soil sample, thereby recovering a xylanase gene from the soil sample.

5. The method according to claim 4, wherein the treated soil sample is a phage library prepared from a soil sample.

6. A polynucleotide probe for isolation or identification of xylanase genes having a sequence which is the same as or fully complementary to at least a portion of any one of Seq. ID Nos. 3 through 22 that is different from the reference xylanase sequence given by Seq. ID. No. 23.

7. A polynucleotide probe for isolation or identification of xylanase genes having a sequence which is the same as or fully complementary to any one of Seq. ID Nos. 3 through 22.