Retrieval of genes and gene fragments from complex samples
AssigneeProkaria ltd.
InventorsGudmundur O. Hreggvidsson, Olafur H. Fridjonsson, Sigurlaug Skirnisdottir, Jakob K. Kristjansson
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Abstract
The present invention features methods of obtaining a specific DNA sequence from a complex sample. The present invention also features methods for obtaining functional genes encoding aminocyclases, amidohydrolases, and/or amylases. In addition, the invention relates to nucleic acid sequence and polypeptide sequences obtained according to the methods of the present invention.
Claims35
A method for obtaining at least one specific DNA sequence related to a target sequence, from a sample comprising a mixed population of a plurality of microbial species, comprising DNA or a mixture of nucleic acids, the method comprising:
- a) extracting the DNA or mixture of nucleic acids from said sample;
- b) hybridizing said DNA or mixture of nucleic acids with a degenerate primer targeted to a single region in said target sequence to synthesize at least one single stranded copy-DNA complementary to a region of said target sequence, said synthesis being primed by said degenerate primer and catalyzed by a DNA-polymerase or a reverse transcriptase;
- and performing a linear amplification of said at least one single stranded copy-DNA by repeated thermal cycling;
- c) purifying the single stranded copy-DNA synthesized in step b);
- d) providing a second primer site to the 3′ end of the single stranded copy-DNA;
- and
- e) amplifying the single stranded copy-DNA using a primer pair wherein a first primer comprises at least a part of the degenerate primer sequence and a second primer which is complementary to the 3′ primer site of step d) or is an arbitrary primer;
- to thereby obtain at least one specific DNA sequence related to said target sequence.
A method for obtaining a functional gene encoding an aminoacylase/amidohydrolase from a sample comprising DNA and/or a mixture of nucleic acids, comprising screening said sample using a nucleic acid probe comprising a nucleotide sequence which is selected from the group consisting of:
- a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31;
- b) a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72;
- c) a nucleotide sequence that encode a polypeptide having at least 75% sequence identity to a polypeptide of step b);
- and
- d) a nucleotide sequence that is complementary to a nucleotide sequences of step a), b), or c).
A method for obtaining a functional gene encoding an amylase from a sample comprising DNA and/or a mixture of nucleic acids, comprising screening said sample using a nucleic acid probe comprising a nucleotide sequence selected from the group consisting of:
- a) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27;
- b) a nucleotide sequence encoding a polypeptide comprising a sequence from the group of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68;
- c) a nucleotide sequence that encodes a polypeptide having at least 65% sequence identity to a polypeptide sequence listed in b);
- and
- d) a nucleotide sequence that is complementary to a sequences of step a), b), c).
A method for obtaining a functional gene encoding an amylase from a sample comprising DNA and/or a mixture of nucleic acids, comprising screening said sample using a nucleic acid probe comprising a nucleotide sequence from the group consisting of SEQ ID NO:
- 19;
- sequences encoding the polypeptide described by SEQ ID NO:60;
- sequences encoding polypeptides having at least 80% sequence identity to SEQ ID NO:60;
- and sequences that are complementary to any of said sequences.
An isolated nucleic acid molecule having a nucleic acid sequence which is part of a gene encoding for an aminoacylase/amidohydrolase, selected from the group consisting of:
- a) SEQ ID NO:1 and SEQ ID NO:2;
- SEQ ID NO:3;
- SEQ ID NO:4;
- SEQ ID NO:5;
- SEQ ID NO:6;
- SEQ ID NO:7;
- SEQ ID NO:8;
- SEQ ID NO:9;
- SEQ ID NO:29;
- and SEQ ID NO:30;
- b) sequences encoding a polypeptide comprising a sequence from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70, and SEQ ID NO:71;
- c) and sequences encoding polypeptides having at least 65% sequence identity with a polypeptide encoded by any of said sequences;
- and
- d) sequences that are complementary to any of said nucleotide sequences of a)-c).
An isolated nucleic acid molecule having a nucleic acid sequence which is part of a gene encoding an aminoacylase/amidohydrolase, selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:31;
- and sequences encoding polypeptides having at least 75% sequence identity with a sequence from SEQ ID NO:69 and SEQ ID NO:72.
An isolated nucleic acid molecule having a nucleic acid sequence which is part of a gene encoding for an amylase, said sequence selected from the group consisting of:
- a) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27;
- b) sequences encoding a polypeptide comprising a sequence from the group of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68;
- c) sequences encoding for polypeptides having at least 65% sequence identity to a polypeptide sequence listed in b);
- and
- d) sequences that are complementary to any of said sequences of a)-c).
An isolated nucleic acid sequence which sequence is part of a gene encoding for an amylase, said sequence from the group consisting of SEQ ID NO:19;
- and sequences encoding for the polypeptide described by SEQ ID NO:
- 60;
- and sequences encoding for polypeptides having at least 80% sequence identity to SEQ ID NO:60.
Dependencies are inferred from the claim text.