Screening Method for Identifying Pufa Pks in Samples

• US 20070259355A1
• Filed: 04/08/2005
• Published: 11/08/2007
• Est. Priority Date: 04/08/2004
• Status: Active Grant

• Current Assignee: Nutrinova Nutrition Specialties & Food Ingredients GmbH (Celanese Corporation)
• Original Assignee: Nutrinova Nutrition Specialties & Food Ingredients GmbH (Celanese Corporation)
• Inventors: Rusing, Matthias, Luy, Markus
• Granted Patent: US 8,409,798 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The invention relates to a method for the rapid and simple identification of PUFA PKS in microorganisms. Said method is characterized by the fact that DNA sections representing PKS that produce specifically for PUFA (polyunsaturated fatty acids) are reproduced in vitro. The PUFA PKS-specific amino acid sequence LGIDSIKRVEIL makes it possible to derive oligonucleotides which are used for the efficient PCR screening for PUFA PKS genes or PUFA-producing microorganisms. The inventive method is particularly suitable for the high throughput screening of microorganisms for PUFA PKS genes.

Claims

1. A method for the demonstration of PUFA-PKS gene-specific nucleic acid sequences in samples, in which

(a) A polymerase chain reaction (PCR) is performed using specific oligonucleotides and with a small part of the sample as a template, and

(b) Obtained PCR products are sequenced and the obtained sequence information is compared with databanks in order to identify new PUFA-PKS sequence information by partial agreement with already known sequences, characterized in that the oligonucleotides used are derived from the amino acid sequence LGIDSIKRVEIL.

2. The method according to claim 1, characterized in that the oligonucleotides are degenerated.

3. The method according to claim 1, characterized in that the length of the oligonucleotides is 10-36 bp.

4. The method according to claim 1, characterized in that the amount of oligonucleotides used is preferably approximately 20 pmol.

5. The method according to claim 1, characterized in that the annealing temperature is approximately 45-65°

C.

6. The method according to claim 1, characterized in that the number of cycles is approximately 20-40.

7. The method according to claim 1, characterized in that nucleic acids isolated from the biomass or the biomass itself being used as template for the PCR.

16. A microorganism containing a nucleic acid identified by the method of claim 1.

8. The amino acid sequence LGIDSIKRVEIL represented in SEQ ID No. 5.

9. Nucleic acid sequences derivable from SEQ ID No. 5.

10. A hybridization probe comprising the nucleic acid according to claim 9.

12. A microorganism contained a nucleic acid according to claim 9.

11. (canceled)

13. Nucleic acid sequence derivable from SEQ ID No. 5 and being selected from the group consisting of the nucleic acids represented in SEQ ID Nos. 6, 7, and 8.

14. A hybridization probe comprising the nucleic acid according to claim 13.

15. A microorganism containing a nucleic acid according to claim 13.