Screening methods and libraries of trace amounts of DNA from uncultivated microorganisms
The invention provides methods for making a gene library from trace amounts of DNA derived from a plurality of species of organisms comprising obtaining trace amounts of cDNA, gDNA, or genomic DNA fragments from a plurality of species of organisms, amplifying the DNA so obtained, and ligating the DNA to a DNA vector to generate a library of constructs in which genes are contained in the DNA. The invention also provides methods for screening clones having DNA recovered from trace amounts of DNA derived from a plurality of species of uncultivated organisms. The invention also provides methods for identifying and enriching for a polynucleotide encoding an activity of interest.
1 . A method for amplifying a DNA template from trace amounts of DNA derived from at least one species of organism comprising:
a) obtaining trace amounts of cDNA, gDNA, or genomic DNA fragments from at least one species of organism;
b) preparing a template from said cDNA, gDNA, or genomic DNA fragments; and
c) amplifying the template.
2 . The method of claim 1 , wherein said template is fragmented.
3 . The method of claim 1 , wherein said trace amounts of cDNA, gDNA, or genomic DNA fragments are partially or completely digested.
4 . The method of claim 2 , wherein the template fragmentation is achieved by enzymatic, chemical, photometric, mechanical or any means that provides segments.
5 . The method of claim 4 , wherein the enzymatic fragmentation comprises use of a DNase or a restriction enzyme.
6 . The method of claim 4 , further comprising filling DNA ends by polymerase extension.
7 . The method of claim 1 , wherein said template is diluted to a degree sufficient to obtain substantially self-ligated products in the presence of ligase and ligase buffer.
8 . The method of claim 1 , wherein said template of step b) is circular.
9 . The method of claim 7 , wherein said substantially self-ligated products are used in said amplifying step.
10 . The method of claim 1 , wherein said amplifying step uses a polymerase.
11 . The method of claim 10 , wherein said polymerase is phi29 polymerase.
12 . The method of claim 4 , wherein the mechanical means comprises use of a shearing means.
13 . The method of claim 1 , wherein the organism comprises uncultured organism.
14 . The method of claim 1 , wherein the at least one organism is derived from an environmental sample
15 . The method of claim 1 , wherein the at least one organism is derived from a contaminated environmental sample.
16 . The method of claim 1 , wherein the organisms comprise a mixture of terrestrial microorganisms or marine microorganisms, or a mixture of terrestrial microorganisms and marine microorganisms.
17 . The method of claim 1 , wherein the organism is an extremophile.
18 . The method of claim 5 , wherein the extremophile comprises one or more organisms selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, psychrotrophs, halophiles, alkalophiles, and acidophiles.
19 . The method of claim 1 , wherein the cDNA or genomic fragments comprise at least an operon, or portions thereof, of the donor microorganisms.
20 . The method of claim 7 , wherein the operon encodes a complete or partial metabolic pathway.
21 . The method of claim 1 , wherein said amplifying step is repeated.
22 . A method for amplifying a DNA template from trace amounts of DNA derived from at least one species of organism comprising:
a) obtaining trace amounts of cDNA, gDNA, or genomic DNA fragments from at least one species of organisms;
b) preparing a circular template from said cDNA, gDNA, or genomic DNA fragments; and
c) amplifying the template.
23 . A method for making a DNA template from trace amounts of DNA isolated from trace amounts of DNA from a mixed population of uncultivated cells comprising:
a) encapsulating individually, in a microenvironment, a plurality of cells from a mixed population of uncultivated cells;
b) creating a template from said cDNA, gDNA, or genomic DNA fragments; and
c) amplifying the template.
24 . The method of claim 23 , wherein said template is fragmented.
25 . The method of claim 23 , wherein said trace amounts of cDNA, gDNA, or genomic DNA fragments are partially or completely digested.
26 . The method of claim 23 , wherein the template fragmentation is achieved by enzymatic, chemical, photometric, mechanical or any means that provides segments.
27 . The method of claim 22 , wherein the enzymatic fragmentation comprises use of a DNAse or a restriction enzyme.
28 . The method of claim 26 , further comprising filling DNA ends by polymerase extension.
29 . The method of claim 23 , wherein said template is diluted to a degree sufficient to obtain substantially self-ligated products in the presence of ligase and ligase buffer.
30 . The method of claim 29 , wherein said substantially self-ligated products are used in said amplifying step.
31 . The method of claim 20 , wherein said amplifying step uses a polymerase.
32 . The method of claim 31 , wherein said polymerase is phi29 polymerase.
33 . The method of claim 26 , wherein the mechanical means comprises use of a shearing means.
34 . The method of claim 23 , wherein the organism is derived from an environmental sample.
35 . The method of claim 23 , wherein the organism is derived from a contaminated environmental sample.
36 . The method of claim 20 , wherein the organism is an extremophile.
37 . The method of claim 31 , wherein the extremophile comprises one or more organisms selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, psychrotrophs, halophiles, alkalophiles, and acidophiles.
38 . The method claim 23 , wherein said microenvironment has trace amounts of cells from at least one species of organism.
39 . The method of claims 1 , 22 , or 23 , wherein said amplifying step is performed by polymerase amplification.
40 . The method of claim 39 , wherein said amplifying step is performed by multiple displacement amplification (MDA).